Microbial consortium and uses thereof

ABSTRACT

Provided herein are, inter alia, microbial compositions and methods of using the same. The microbial compositions provided include, inter alia, therapeutically effective amounts of  Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus  and  Pediococcus pentosaceus  and are particularly useful for methods of treating and preventing inflammatory diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International Application No. PCT/US2017/020809, filed Mar. 3, 2017, which claims the benefit of priority to U.S. Provisional Application No. 62/304,087, filed Mar. 4, 2016, the entire contents of each of which are hereby incorporated by reference in their entireties and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under grant numbers R21 AT004732, P01 AI089473, and HL080074 awarded by the National Institutes of Health. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The content of the text file named “48536_575C01US_Sequence_Listing.txt”, which was created on Apr. 5, 2018, and is 107,578 bytes in size, is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Recent studies provide evidence that microbial communities residing in the human gut play a key role in the development and modulation of the host immune response. For instance, the presence of particular Clostridium species has been shown to induce specific T-cell repertoires [Atarashi, et al. (2011) Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331(6015):337-341]. Despite the complexity of the gut microbiome, the presence or absence of specific bacterial species can dramatically alter the adaptive immune environment.

BRIEF SUMMARY OF THE INVENTION

Provided herein are novel methods and microbial compositions including Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and Pediococcus penrtosaceus, which are surprisingly useful for the treatment of dysbiosis, infections, and inflammatory diseases.

An aspect provides methods and compositions comprising a bacterial population that comprises, consists essentially of, or consists of, 1, 2, 3, 4, 5, 6, 7, or 8 (or at least 1, 2, 3, 4, 5, 6, 7, or 8) bacterial species. In embodiments, the bacterial population comprises, consists essentially of, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cysobacter sp., Pediococcus sp., Bifidobacterium sp., and Clostridium sp. In embodiments, the bacterial population comprises Lactobacillus sp. and Faecalibacterium prausnitzii. In embodiments, the bacterial population comprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments, the bacterial population comprises Lactobacillus sp., and Myxococcus xanthus. In embodiments, the bacterial population comprises Lactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterial population comprises Lactobacillus sp. and Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, the bacterial population comprises Lactobacillus sp. and Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. In embodiments, the bacterial population comprises Lactobacillus sp. and Clostridium hiranonis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii. In embodiments, the bacterial population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination of the following: Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii. Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, and Pediococcus parvulus. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus.

In an aspect, a method for administering isolated bacteria is provided. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect, a method of bacterial supplementation is provided. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect, a method of treating or preventing inflammation in a subject in need thereof is provided. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect, a microbial composition is provided. In embodiments, the microbial composition comprises Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the microbial composition comprises Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Pediococcus pentosaceus and a biological carrier suitable for administration to the gut. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect, a microbial composition is provided. In embodiments, the microbial composition comprises Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the microbial composition comprises Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus and a biological carrier suitable for administration to the gut. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect a pharmaceutical composition is provided. In embodiments, the pharmaceutical composition comprises Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the pharmaceutical composition comprises Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the pharmaceutical composition includes a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and Pediococcus pentosaceus and a pharmaceutically acceptable excipient is provided. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect a method of treating or preventing an inflammatory disease in a subject in need thereof is provided. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. The method includes administering to the subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect is provided a method of increasing an anti-inflammatory metabolite in a subject in need thereof is provided. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the method includes administering to the subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect is provided a method of decreasing a pro-inflammatory metabolite in a subject in need thereof is provided. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments, the method comprises administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. In embodiments, the method includes administering to the subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect a method of detecting an anti-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease is provided. The method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of an anti-inflammatory metabolite in the biological sample.

In an aspect a method of detecting a pro-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease is provided. The method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of a pro-inflammatory metabolite in the biological sample.

In an aspect, a method of determining whether a subject has or is at risk of developing an inflammatory disease is provided. The method includes (i) detecting an expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an pro-inflammatory metabolite or a decreased expression level of an anti-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In an aspect, a method of determining whether a subject has or is at risk of developing an inflammatory disease is provided. The method includes (i) detecting an expression level of one or more pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein an increased expression level of an pro-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In an aspect, a method of determining whether a subject has or is at risk of developing an inflammatory disease is provided. The method includes (i) detecting an expression level of one or more anti-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein a decreased expression level of an anti-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In an aspect, a method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy is provided. The method includes (i) determining a first expression level of an anti-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of an anti-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of an anti-inflammatory metabolite to the first expression level of an anti-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject.

In an aspect, a method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy is provided. The method includes (i) determining a first expression level of a pro-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of a pro-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of a pro-inflammatory metabolite to the first expression level of a pro-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject.

In an aspect, a method of determining an inflammatory disease activity in a subject is provided. The method includes (i) detecting an expression level of one or more anti-inflammatory metabolites in a subject; (ii) determining whether the expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in the subject; and (iii) based at least in part on the expression level in step (ii), determining the inflammatory disease activity in the subject.

In an aspect, a method of determining an inflammatory disease activity in a subject is provided. The method includes (i) detecting an expression level of one or more pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in the subject; and (iii) based at least in part on the expression level in step (ii), determining the inflammatory disease activity in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Significantly improved lung histology and decreased goblet cell hyperplasia is evident only in mice supplemented with C+Lj. Histological samples from three mice in each of the six experimental groups were stained to visualize goblet cell hyperplasia in duplicate studies. FIG. 1A: Representative images of PAS staining for each of the six groups clearly show that mucin secretion of goblet cells (stained) is induced in CRA challenged mice and that supplementation with L. johnsonii and microbial consortium is the only treatment group that protects against this induction. FIG. 1B: Image J was used to quantify the percentage of area in each image that is positive for PAS staining. Each data point is represented by an individual symbol generated in two independent murine studies. Statistical analyses of this data show significant reductions in the percentage of PAS staining associated with the C+Lj consortium supplemented mice compared to all other CRA treated groups.

FIG. 2. Significantly decreased expression of MUC5AC in mice supplemented with C+Lj supports histological findings. RNA extracted from the lung of each animal was used to examine the gene expression level of MUC5AC, a gene involved in mucin production by goblet cells. Data from two independent murine studies is presented. Statistical analyses of this data show significant reductions in the percentage of Muc5ac gene expression associated with C+Lj supplemented mice compared to all other CRA treated groups.

FIGS. 3A-3C. Supplementation with C+Lj resulted in decreased airway expression of cytokines associated with allergic response. RNA extracted from the lung of each animal was also used to examine the gene expression level of multiple cytokines associated with allergic responses, including IL-4 (FIG. 3A), IL-13 (FIG. 3B), IL-10 (FIG. 3C) and IL-17. Similarly, Applicants observed a significant decrease in cytokine expression associated with C+Lj supplementation for the Th2-associated cytokines (IL-4 and IL-13), as well as IL-10. Data from two independent replicate studies is presented.

FIG. 4. Increased percentage of 11-17 secreting T helper cells (CD3⁺CD4⁺) is most significant in mice supplemented with C+Lj. Flow cytometry data from splenocytes reveal a significant increase in the percentage of CD4⁺ cells expressing IL-17, a cytokine associated with Th 17 cells. This observation held true across duplicated studies, and the data from both studies are shown here.

FIGS. 5A-5B. The bacterial consortium including L. johnsonii, but not L. rhamnosus LGG, provides attenuation of allergic sensitization. The CRA allergen mouse study was performed using either L. rhamnosus LGG (LGG) or L. johnsonii (Lj) as the Lactobacillus anchor species included in the bacterial consortium supplement. To evaluate the effect of each consortium on sensitization, the expression level of MUC5AC was determined in lung tissue using qPCR. The L. johnsonii-based consortium significantly decreased MUC5AC expression, whereas the L. rhamnosus LGG consortium failed decrease the expression of this allergic response biomarker.

FIGS. 6A-6C. Fecal water from a non-atopic neonate significantly reduces expression of IL4 and IL13 responses. FIG. 6A: Fecal water exposure significantly decreases the number of CD4+ T-helper 2 cells in one but not both donors. FIG. 6B: IL4 expression is significantly reduced in both donors. FIG. 6C: IL13 expression is significantly reduced in both donors.

FIGS. 7A-7B. Cell-free supernatant from distinct neonatal gut microbiome isolates of Candida consistently induce CD4+ IL4+ (Th2) cells (FIG. 7A). Specific species induce or suppress CD4+ IL10+ (T-reg) cells (FIG. 7B). BHI, sterile brain heart infusion medium exposure (control); CT, C. tropicalis; CP, C. parapsilosis; CO, C. orthopsilosis; and CT, C. tropicalis; Control, non-antigen stimulated conditions; CRA, cockroach stimulated T-cells.

FIGS. 8A-8D. UC microbiotypes exhibit significantly different disease severity and duration. FIG. 8A: Simple colitis disease severity score. FIG. 8B: Number of extra-colonic manifestations. FIG. 8C: Disease duration. FIG. 8D. Number of family members with inflammatory bowel disease (IBD).

FIGS. 9A-9D. In vitro fecal water assays reveal that UC patients exhibit significantly distinct Th2 ratios, IL4 production and CD8+ IL17+ populations. FIG. 9A: A significant skew towards Th2 responses characterizes UC patients compared to healthy controls. FIG. 9B: UC microbiotypes exhibit significant differences in the degree of Th2 skew, with the most severe microbiotype (MBT-1) exhibiting the most profound Th2 skew. FIG. 9C: IL4 expression is significantly different across UC microbiotypes, with the MBT-1 group exhibiting significantly higher IL4 expression compared to the lowest disease severity group. FIG. 9D: MBT-1 patients exhibit significantly greater numbers of CD8+ IL17+ cells compared with the two other lower disease severity groups.

FIG. 10. Experimental timeline illustrating phosphate buffered saline (PBS) or the therapeutic consortium (TC) supplementation regime and CRA challenge schedule in a murine model of airway allergic sensitization.

FIGS. 11A-11B. Oral supplementation of mice with the TC promotes increased relative abundance of genera associated with induction of immune tolerance. FIG. 11A: Microbiome composition was determined in the feces of the animals in the study using 16S rRNA sequencing. Cluster analysis revealed differences in microbiome composition across treatment groups. The TC-supplemented animals showed a significantly distinct composition compared with the control groups. Specifically, TC-supplemented animals were enriched for species with the potential for immunomodulatory activity (e.g., Bifidobacterium, Clostridia species belong to Clade IV and XIV, Lachnospira, and Bacteroides). FIG. 11B: Pie chart showing enriched taxa in CRA-TC treated animals.

FIGS. 12A-12B. Oral supplementation of mice with the TC promotes metabolic reprogramming in both the gut lumen and periphery and includes significant increases in circulating itaconate, which is associated with a repair macrophage effector phenotype. FIG. 12A: Principle components analysis of the dominant luminal metabolites using un-targeted liquid chromatography mass spectrometry revealed distinct metabolic profiles between the three groups (Canberra Distance Matrices; PERMANOVA, R²=0.29, p=0.005). FIG. 12B: Principle components analysis of the circulating metabolites identified in the serum using the same strategy also revealed significant differences (Canberra Distance Matrices; PERMANOVA, R²=0.29, p=0.002 between the groups examined. Untargeted LC GC Mass spectrometry was used to identify and determine the relative concentrations of several hundred metabolites in the feces (FIG. 12A) and serum (FIG. 12B) of mice supplemented with the TC or PBS prior to cockroach (CRA) antigen challenge. Significant spatial separation of TC-supplemented versus PBS supplemented animals on a PcoA plot indicates that the profile of metabolites in the feces and serum of these animals is significantly different.

FIGS. 13A-13B. FIG. 13A: Histological sections of the murine airway (lung) indicate that oral supplementation of mice with the metabolically active therapeutic consortium (CRA+TC) significantly reduces inflammatory influx [Hemotoxylin and Eosin (H&E) Staining; dark stained nucleated cells] in a murine model of airway allergic sensitization. L. johnsonii alone does not confer protection (CRA+Lj), nor does supplementation of animals with four of the TC (omitting L. johnsonii; CRA+C), indicating that L. johnsonii acts in synergy with the other four members of the TC to effect protection at the airway mucosal surface. A heat-killed, metabolically inactive TC also does not confer protection indicating that only the metabolically active TC protects. FIG. 13B: Gene expression analyses of CCL-11 expression, a marker of eosinophils, confirm that the CRA+TC group exhibits significantly reduced eosinophil presence in the lungs following allergic sensitization.

FIG. 14A-14B. FIG. 14A: Histological sections of the murine airway (lung) indicate that oral supplementation of mice with the metabolically active therapeutic consortium (CRA+TC) significantly reduces mucin hyper-secretion (Periodic Acid-Schiff (PAS) Staining: dark staining) in a murine model of airway allergic sensitization. L. johnsonii alone does not reduce mucin secretion (CRA+Lj), nor does supplementation of animals with four of the TC (omitting L. johnsonii; CRA+C), indicating that L. johnsonii acts in synergy with the other four members of the TC to suppress mucin secretion at the airway mucosal surface. A heat-killed, metabolically inactive TC also does not reduce mucin secretion indicating that only the metabolically active TC protects. FIG. 14B: Gene expression analyses of Muc5AC expression, the primary gene responsible for mucin secretion in the airways, confirm that the CRA+TC group exhibits significantly reduced mucin gene expression the lungs, compared to the other treatment groups.

FIG. 15-A-15C. Oral supplementation of mice with the metabolically active TC significantly reduces cytokine expression associated with allergic inflammation in a murine model of airway allergic sensitization. FIG. 15A: Boxplot demonstrating a significant decrease in relative change in expression of IL-13 in animals treated with TC and CRA challenged compared with PBS treatment and CRA challenged. FIG. 15B: Boxplot demonstrating a significant decrease in relative change in expression of IL-4 in animals treated with TC and CRA challenged compared with PBS treatment and CRA challenge. FIG. 15C: Boxplot demonstrating a significant decrease in relative change in expression of IL-10 in animals treated with TC and CRA challenged compared with PBS treatment and CRA challenge.

FIGS. 16A-16F. Oral supplementation of mice with the TC results in a repair macrophage effector phenotype in a murine model of airway allergic sensitization. In CRA+TC treated mice, CD11b^(hi)F4/80^(hi) macrophages form a larger percentage of the non-lymphocyte population in (FIG. 16A) mesenteric lymph nodes, (FIG. 16B) spleen, and (FIG. 16C) lung compared to CRA+PBS treated animals. CRA+TC and CRA+PBS treated animals show similar percentages of CD11b^(hi)F4/80^(hi) CD206⁺ (M2) macrophages in the non-lymphocyte population in both (FIG. 16D) mesenteric lymph nodes and (FIG. 16E) spleen. FIG. 16F: In lung, CRA+TC treated animals show an increased percentage of CD11b^(hi)F4/80^(hi) CD206⁺ (M2) macrophages in the non-lymphocyte population compared to CRA+PBS treated mice.

FIG. 17. Table showing treatment groups utilized in murine model of airway allergic sensitization study.

FIGS. 18A-18C. Metabolic reprogramming in gut lumen following oral supplementation of mice with the therapeutic consortium (TC) promotes increased concentrations of specialized lipids, plasmalogens, which are enriched in polyunsaturated fatty acids (PUFAs). PUFAs are increased in the feces of neonates at low risk for allergies and asthma in childhood. FIG. 18A: Carbohydrate compounds decreased in concentration following treatment with the TC. FIG. 18B: Energy compounds decreased in concentration following treatment with the TC. FIG. 18C: Lipid compounds decreased in concentration (PUFAs, Long chain fatty acids, acyl-glycerols, and branched fatty acids) and increased (phospholipids and plasmalogens) following treatment with the TC.

FIG. 19. Bacterial and fungal α- and β-diversity are related to participant age at the time of fecal-sample collection. Bacterial and fungal α-diversities are inversely correlated (Shannon's index; n=188; Pearson's correlation, r²=−0.24; P<0.001).

FIGS. 20A-20B. Compositionally distinct, age-independent NGM states exist in neonates, exhibit significant differences in fungal taxonomy and are related to the RR of atopy at the age of 2 years. FIG. 20A: NGM participants do not differ significantly in age (n=130; Kruskal-Wallis; P=0.256). Box plots are defined by the 25^(th) and 75^(th) percentiles. Center line represents the median (50^(th) percentile). Whiskers are defined as 1.5 times the interquartile range (IQR, 75^(th)-25^(th) percentile), plus or minus the 75^(th) and 25^(th) percentiles, respectively. FIG. 20B: The sum of allergen-specific serum IgE concentrations measured at 2 years of age (n=130) is significantly higher for NGM3 compared with NGM1 participants (Welch's t test; P=0.034). Box plots are constructed as defined in FIG. 20A.

FIG. 21. NGMs exhibit significantly different RRs of PM atopy development at age 2 years and of parental report of doctor-diagnosed asthma at age 4 years. Significance of risk ratios between microbiota states was calculated on the basis of log-binomial regression.

FIGS. 22A-22F. Sterile fecal water from NGM3 participants induces CD4⁺ cell population dysfunction associated with atopic asthma. Dendritic cells and autologously purified naïve CD4⁺ cells from the serum of two healthy adult donors (biological replicates) were incubated with sterile fecal water from NGM1 (n=7; three biological replicates per sample) or NGM3 (n=5; three biological replicates per sample) participants. FIGS. 22A and 22B: Fecal water from NGM3 participants induced significantly increased proportions of CD4⁺IL-4⁺ cells (LME, P<0.001; center line represents mean) (FIG. 22A) and expression of IL-4 (LME; P=0.045) (FIG. 22B). FIG. 22C: Fecal water from both NGM1 and NGM3 participants induced significantly increased proportions of CD4⁺CD25⁺FOXP3⁺ cells (LME; P<0.001 for NGM1 and P=0.017 for NGM3), compared with control. FIG. 22D: Weighted correlation network analysis identified a metabolic module that differentiates NGM3 from NGM2 and NGM1 participants (n=28; ANOVA; P=0.038). Box plots define the 25^(th) and 75^(th) percentiles; the median is represented by the center line. IQR (75^(th)-25^(th) percentile) is represented by whiskers. FIG. 22E: Scatterplot of metabolite significance versus module membership (MM) of the 12 metabolites in the NGM3-discriminating metabolic module. Metabolites with a higher metabolite significance value discriminate NGM3 from other NGMs. Metabolites plotted above the dashed line (representing the overall p-value for between-NGM differences) are significantly associated with NGM differentiation (P<0.05), and were detected in higher concentrations in NGM3 compared to the other NGMs. MM values indicate the degree of interconnectedness of a specific metabolite to other metabolites in the module (higher MM value indicates greater interconnectedness). FIG. 22F: When the same ex vivo assay that was performed in FIGS. 22A-22C was used, 12,13-DiHOME significantly reduced the proportion of CD4⁺CD25⁺FOXP3⁺ cells at three different concentrations compared to vehicle control (LME; P=0.04, P<0.001, P=0.001 for concentrations of 75, 130 and 200 μM, respectively; center line represents mean proportion of cells).

FIG. 23. Dirichlet multinomial mixture model identifies three compositionally distinct bacterial NGMs as the best model fit. Model fit was based on the Laplace approximation to the negative log model where a lower value indicates a better model fit.

FIG. 24. Sterile fecal water from NGM3 participants induces a CD4⁺IL-4⁺ cell skew. Dendritic cells from serum of two healthy adult donors (biological replicates), were incubated with sterile fecal water from NGM1 (n=7; three biological replicates per sample) or NGM3 (n=5; three biological replicates per sample) participants, prior to co-incubation with autologously purified naïve CD4⁺ cells. NGM3 fecal water induces a trend toward a CD4⁺IL-4⁺ cell skew compared with NGM1 (LME; P=0.095).

FIG. 25. Confirmation that the concentration of the dihydroxy fatty acid 12, 13 DiHOME, is significantly increased in the NGM3 sample subset used for ex vivo assays. Using the subset of samples employed in the ex vivo DC-T-cell assay and based on metabolite scaled intensity data, 12, 13 DiHOME is significantly increased in relative concentration in NGM3 (n=7) compared to NGM1 (n=5) samples (Welch's-test; P=0.033).

FIG. 26. Allergens used to determine PM atopy status of participants in this study. Mean and median of allergen-specific IgE (IU ml⁻¹) is provided for each.

FIG. 27. Risk ratio of IGMs (infants>6 months old) for developing atopy or having parental report of doctor's diagnosis of asthma. Risk ratios were calculated based on log-binomial regression.

FIG. 28. Fungal taxa exhibiting significantly increased relative abundance in lower-risk NGM1 versus higher-risk NGM3 neonatal gut microbiota. Significant difference in relative abundance was determined using a zero-inflated negative binomial regression model (q<0.20). White background indicates taxa enriched in NGM1 (compared with NGM3), gray background indicates taxa enriched in NGM3 (compared with NGM1). Findings are ranked by difference in relative abundance (NGM1-NGM3).

FIG. 29. Fungal taxa exhibiting significantly increased relative abundance in lower-risk NGM2 versus higher-risk NGM3 neonatal gut microbiota. Significant difference in relative abundance was determined using zero-inflated negative binomial regression model (q<0.20). White background indicates taxa enriched in NGM2 (compared with NGM3), gray background indicates taxa enriched in NGM3 (compared with NGM2). Findings are ranked by difference in relative abundance (NGM2-NGM3).

FIG. 30. Procrustes analyses of 16S rRNA-based β-diversity, PICRUSt and metabolomic datasets. Results from Procrustes analyses indicate that bacterial β-diversity, PICRUSt and metabolomic data is highly and significantly correlated.

FIGS. 31A-31C. Comparison of healthy (n=13) and UC-associated (n=30) fecal microbiotas. FIG. 31A: Bacterial diversity. Horizontal bars represent means±standard deviations. P values were obtained by two-tailed Student t test. FIG. 31B: Bacterial community composition represented by nonmetric multidimensional scaling (NMDS) of pairwise weighted UniFrac distances. FIG. 31C: Bacterial community composition of UC patients stratified by ethnicity (18 EU UC, 12 SA UC) represented by NMDS of pairwise weighted UniFrac distances. In FIG. 31B and FIG. 31C, each dashed ellipse represents the 95% confidence interval for the centroid of each stratification group as calculated by ordiellipse.

FIGS. 32A-32D. Clinical measurements of UC severity among UC MCSs (11 for MCS1, 8 for MCS2, 4 for MCS3, 3 for MCS4). FIG. 32A: Simple clinical colitis activity. FIG. 32B: Number of extracolonic symptoms. FIG. 32C: Number of family members diagnosed with IBD. FIG. 32D: Duration of disease. All pairwise comparisons were done with a two-tailed Dunn test. Only P values of <0.1 are indicated. EU UC, squares; SA UC, circles.

FIG. 33A-33K. In vitro human T-cell activity following coculture with autologous DCs coincubated with sterile fecal water. FIG. 33A: Th1-to-Th2 ratio; FIG. 33B: Th1 frequency; FIG. 33C: Th2 frequency; FIG. 33D: Th17 frequency; e, regulatory T-cell frequency (48 healthy, 116 UC). Comparisons of the Th1 frequencies (FIG. 33F), Th2 frequencies (FIG. 33G), and Th1-to-Th2 ratios (FIG. 33H) of healthy and UC MCSs are shown (48 for healthy, 48 for MCS1, 40 for MCS2, 16 for MCS3, and 8 for MCS4). Concentrations of IL-4 (FIG. 33I), IL-5 (FIG. 33J), and IL-13 (FIG. 33K) in cell supernatant following coculture of human T cells with autologous DCs challenged with sterilized fecal water from healthy participants and MCS1 and MCS2 patients are shown (48 for healthy participants, 48 for MCS1 patients, and 40 for MCS2 patients). Data were generated from four (FIGS. 33A-33H) or two (FIG. 33I-33K) replicate experiments with DCs/T cells obtained from two anonymous PBMC donors. Horizontal bars (mean fitted values for each group) and P values were determined by linear mixed-effect modeling (see Materials and Methods). P values of <0.1 are indicated.

FIGS. 34A-34H. Comparison of healthy (n=13) and UC-associated (n=30) fecal fungal microbiotas. FIG. 34A: Fungal α diversity stratified by healthy status. FIG. 34B: Fungal community composition represented by NMDS of pairwise Bray-Curtis distances. Participants are colored by health status. Bacterial α diversity FIG. 34C and fungal α diversity FIG. 34D were stratified by health status and ethnicity (10 healthy EU, 3 healthy SA, 18 UC EU, 12 UC SA). FIG. 34E: Simple clinical colitis activity of UC patients stratified by ethnicity (14 EU UC, 12 SA UC). P values were obtained by two-tailed rank sum test. FIG. 34F Bacterial community composition of all participants stratified by ethnicity (28 EU, 15 SA) represented by NMDS of pairwise weighted UniFrac distances. FIG. 34G: Fungal community composition of all participants stratified by ethnicity (28 EU, 15 SA) represented by NMDS of pairwise Bray-Curtis distances. FIG. 34H: PhyloChip-profiled bacterial community composition of UC patients stratified by ethnicity (15 EU UC, 11 SA UC) represented by NMDS of pairwise Canberra distances. In panels a, c, and d, horizontal bars represent means±standard deviations. P values were obtained by two-tailed t test. In FIG. 34B and FIGS. 34F-34H, each dashed ellipse represents the 95% confidence interval for the centroid of each participant stratification group as calculated by ordiellipse. Each dot/square represents a single fecal sample obtained from a single donor.

FIGS. 35A-35B. Bacterial community compositions of UC patients stratified by UC MCS. FIG. 35A: NMDS of pairwise weighted UniFrac distances for 16S rRNA profiles obtained via Illumina MiSeq (12 MCS1, 10 MCS2, 4 MCS3, 3 MCS4, 1 other). FIG. 35B: NMDS of pairwise Canberra distances for 16S rRNA profiles obtained via PhyloChip (10 MCS1, 8 MCS2, 4 MCS3, 2 MCS4, 1 other). Each dashed ellipse represents the 95% confidence interval for the centroid of each participant stratification group as calculated by ordiellipse. Each dot/square represents a single fecal sample obtained from a single donor.

FIG. 36. In vitro human T-cell activity following coculture with autologous DCs coincubated with sterile fecal water. Induced Th1-to-Th2 ratios of EU UC (n=) and SA UC patients are compared. Data were generated from four replicate experiments with DCs/T cells obtained from two anonymous PBMC donors. Horizontal bars (mean fitted values for each group) and P values were determined by linear mixed-effect modeling.

FIG. 37. Breakdown of Study Participant Cohort. Note: one SA-UC participant failed to report their sex.

FIG. 38. Description of Metabolon QC Samples.

FIG. 39. Metabolon QC Standards.

DETAILED DESCRIPTION I. Definitions

While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

The term “exogenous” refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an “exogenous promoter” as referred to herein is a promoter that does not originate from the plant it is expressed by. Conversely, the term “endogenous” or “endogenous promoter” refers to a molecule or substance that is native to, or originates within, a given cell or organism.

The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

The term “isolated”, when applied to a bacterium, refers to a bacterium that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man, e.g. using artificial culture conditions such as (but not limited to) culturing on a plate and/or in a fermenter. Isolated bacteria include those bacteria that are cultured, even if such cultures are not monocultures. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In embodiments, isolated bacteria are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%6, or more than about 99% pure. In embodiments, a bacterial population provided herein comprises isolated bacteria. In embodiments, a composition provided herein comprises isolated bacteria. In embodiments, the bacteria that are administered are isolated bacteria.

As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified”, when applied to a bacterium, refer to a bacterium that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A bacterium or a bacterial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the bacterium or bacterial population, or by passage through culture, and a purified bacterium or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified bacteria and bacterial populations are more than about 80%, about 85%, about 90%0, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In the instance of microbial compositions provided herein, the one or more bacterial types (species or strains) present in the composition can be independently purified from one or more other bacteria produced and/or present in the material or environment containing the bacterial type. Microbial compositions and the bacterial components thereof are generally purified from residual habitat products.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes (e.g. cyanine), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

“Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules and/or cells such as bacterial cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, a resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, an antibody domain as described herein and an antibody-binding domain. In embodiments contacting includes, for example, allowing an antibody domain as described herein to interact with an antibody-binding domain.

“Patient” or “subject in need thereof” refers to a living member of the animal kingdom suffering from or that may suffer from the indicated disorder. In embodiments, the subject is a member of a species comprising individuals who naturally suffer from the disease. In embodiments, the subject is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In embodiments, the subject is a human. In embodiments, the subject is a non-mammalian animal such as a turkey, a duck, or a chicken. In embodiments, a subject is a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein.

The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In embodiments, the disease is an inflammatory disease (e.g. asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, erythema nodosum, or any other inflammatory disease mentioned herein). As used herein, a “symptom” of a disease includes any clinical or laboratory manifestation associated with the disease, and is not limited to what a subject can feel or observe.

As used herein, the term “inflammatory disease” refers to a disease or condition characterized by aberrant inflammation (e.g., an increased level of inflammation compared to a control such as a healthy person not suffering from a disease). Non-limiting examples of inflammatory diseases include allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

As used herein the term “dysbiosis” means a difference in the gastrointestinal microbiota compared to a healthy or general population. In embodiments, dysbiosis comprises a difference in gastrointestinal microbiota commensal species diversity compared to a healthy or general population. In an embodiment, dysbiosis comprises a decrease of beneficial microorganisms and/or increase of pathobionts (pathogenic or potentially pathogenic microorganisms) and/or decrease of overall microbiota species diversity. Many factors can harm the beneficial members of the intestinal microbiota leading to dysbiosis, including (but not limited to) antibiotic use, psychological and physical stress, radiation, and dietary changes. In an embodiment, dysbiosis comprises or promotes the overgrowth of a bacterial opportunistic pathogen such as Enterococcus faecalis, Enterococcus faecium, or Clostridium difficile. In an embodiment, the dysbiosis comprises a reduced amount (absolute number or proportion of the total microbial population) of bacterial or fungal cells of a species or genus (e.g., 5%, 10%, 15%, 20%¹, 25%, 30%, 35%, 40%, 45%/O, 50%, 55%, 600, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more lower) compared to a healthy subject (e.g., a corresponding subject who does not have an inflammatory disease, an infection, and who has not been administered an antibiotic within about 1, 2, 3, 4, 5, or 6 months, and/or compared to a healthy or general population). In an embodiment, the dysbiosis comprises an increased amount (absolute number or proportion of the total microbial population) of bacterial or fungal cells within a species or genus (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%/O or more higher) compared to a healthy subject (e.g., a corresponding subject who does not have an inflammatory disease, an infection, and who has not been administered an antibiotic within about 1, 2, 3, 4, 5, or 6 months, and/or compared to a healthy or general population). In an embodiment, a subject who comprises a gastrointestinal infection, gastrointestinal inflammation, diarrhea, colitis, or who has received an antibiotic within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks is deemed to comprise dysbiosis. In an embodiment, the impaired microbiota comprises small intestinal bacterial or fungal overgrowth. Antibiotic administration (e.g., systemically, such as by intravenous injection or orally) is a common and significant cause of major alterations in the normal microbiota. Thus, as used herein, the term “antibiotic-induced dysbiosis” refers to dysbiosis caused by or following the administration of an antibiotic.

Non-limiting examples of dysbiosis are described in the examples provided herein. Non-limiting examples of dysbiosis in the context of neonates are also described in Fujimura et al. (2016) “Neonatal gut microbiota associates with childhood multisensitized atopy and T cell differentiation” Nature Medicine 22(10): 1187-1191 (hereinafter “Fujimura et al. 2016”), the entire content of which (including all supplemental information and data) is incorporated herein by reference. In some embodiments, a subject with dysbiosis has the NGM3 microbiome profile as set forth in Fujimura et al. 2016. Non-limiting examples of dysbiosis in the context of ulcerative colitis are described in Mar et al. (2016) “Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients” mBio 7(4):e01072-16 (herein after “Mar et al. 2016”), the entire content of which (including all supplemental information and data) is incorporated herein by reference. In some embodiments, a subject with dysbiosis has the MCS4 microbiome profile as set forth in Mar et al. 2016. In some embodiments, a subject with dysbiosis has the MCS 3 microbiome profile as set forth in Mar et al. 2016. In some embodiments, a subject with dysbiosis has the MCS2 microbiome profile as set forth in Mar et al. 2016. In some embodiments, a subject with dysbiosis has the MCS1 microbiome profile as set forth in Mar et al. 2016.

The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., an allergy, asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum) means that the disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.

The term “aberrant” as used herein refers to different from normal. When used to describe enzymatic activity, aberrant refers to activity that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease-associated amount (e.g. by using a method as described herein), results in reduction of the disease or one or more disease symptoms.

A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. dysbiosis, an autoimmune disease, inflammatory autoimmune disease, cancer, infectious disease, immune disease, or other disease) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. microbiome, RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophage-like synoviocytes, etc).

One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

The term “diagnosis” refers to a relative probability that a disease (e.g. an autoimmune, inflammatory autoimmune, cancer, infectious, immune, or other disease) is present in the subject. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject with respect to a disease state. For example, in the context of the present invention, prognosis can refer to the likelihood that an individual will develop a disease (e.g. an autoimmune, inflammatory autoimmune, cancer, infectious, immune, or other disease), or the likely severity of the disease (e.g., duration of disease). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.

“Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), feces and feces fractions or products (e.g., fecal water, such as but not limited to fecal water separated from other fecal components and solids by methods such as centrifugation and filtration) sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, dendritic cells, T-cells, etc. In embodiments, a sample is obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

A “cell” as used herein, refers to a cell carrying out metabolic or other functions sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

As used herein the abbreviation “sp.” for species means at least one species (e.g., 1, 2, 3, 4, 5, or more species) of the indicated genus. The abbreviation “spp.” for species means 2 or more species (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the indicated genus. In embodiments, methods and compositions provided herein comprise a single species within an indicated genus or indicated genera, or 2 or more (e.g., a plurality comprising more than 2) species within an indicated genus or indicated genera. In embodiments, 1, 2, 3, 4, 5, or more or all or the indicated species is or are isolated. In embodiments, the indicated species are administered together. In embodiments, each of the indicated species is present in a single composition that comprises each of the species. In embodiments, each of the species is administered concurrently, e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, or 60, 1-5, 1-10, 1-30, 1-60, or 5-15 seconds or minutes of each other.

In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like. “Consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified.

As used herein, the term “about” in the context of a numerical value or range means±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

In the descriptions herein and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.

As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise.

II. Bacterial Populations and Microbial Compositions

In an aspect, a composition comprising a bacterial population that comprises, consists essentially of, or consists of, 1, 2, 3, 4, 5, 6, 7, or 8 (or at least 1, 2, 3, 4, 5, 6, 7, or 8) bacterial species. In embodiments, the bacterial population comprises, consists essentially of, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp., Pediococcus sp., Bifidobacterium sp., and Clostridium sp. In embodiments, the bacterial population comprises Lactobacillus sp. and Faecalibacterium prausnitzii. In embodiments, the bacterial population comprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments, the bacterial population comprises Lactobacillus sp., and Myxococcus xanthus. In embodiments, the bacterial population comprises Lactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterial population comprises Lactobacillus sp. and Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, the bacterial population comprises Lactobacillus sp. and Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. In embodiments, the bacterial population comprises Lactobacillus sp. and Clostridium hiranonis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii. In embodiments, the bacterial population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination of the following: Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, and Pediococcus parvulus. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In embodiments, the bacteria are isolated bacteria.

In an aspect, a composition including Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp is provided. In embodiments, (i) the Lactobacillus sp. is Lactobacillus johnsonii; (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. is Pediococcus pentosaceus.

In an aspect, a composition including Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and Pediococcus sp is provided. In embodiments, (i) the Lactobacillus sp. is Lactobacillus johnsonii; (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Cystobacter sp. is Cystobacter fuscus; and (v) the Pediococcus sp. is Pediococcus pentosaceus.

In embodiments, the bacterial population further comprises Bifidobacterium sp. or Clostridium sp. In embodiments, the Bifidobacterium sp. is Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. In embodiments, the Clostridium sp. is Clostridium hiranonis.

In an aspect, a microbial composition is provided. The composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Pediococcus pentosaceus and a biological carrier suitable for administration to the gut.

In an aspect, a microbial composition is provided. The composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus and a biological carrier suitable for administration to the gut.

In embodiments, the biological carrier is suitable for oral or rectal administration. In embodiments, the biological carrier is suitable for colonization of the gut. A “biologically acceptable” (or “pharmacologically acceptable”) carrier as referred to herein refers to molecular entities and compositions as described herein that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human.

In embodiments, the composition includes less than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 different species of bacteria. In embodiments, the composition includes less than about 20 different species of bacteria. In embodiments, the composition includes less than 20 different species of bacteria. In embodiments, the composition includes less than about 15 different species of bacteria. In embodiments, the composition includes less than 15 different species of bacteria. In embodiments, the composition includes less than about 10 different species of bacteria. In embodiments, the composition includes less than 10 different species of bacteria. In embodiments, the composition includes less than about 9 different species of bacteria. In embodiments, the composition includes less than 9 different species of bacteria. In embodiments, the composition includes less than about 8 different species of bacteria. In embodiments, the composition includes less than 8 different species of bacteria. In embodiments, the composition includes less than about 7 different species of bacteria. In embodiments, the composition includes less than 7 different species of bacteria. In embodiments, the composition includes less than about 6 different species of bacteria. In embodiments, the composition includes less than 6 different species of bacteria. In embodiments, the composition includes less than about 5 different species of bacteria. In embodiments, the composition includes less than 5 different species of bacteria. In embodiments, the composition includes less than about 4 different species of bacteria. In embodiments, the composition includes less than 4 different species of bacteria. In embodiments, the composition includes less than about 3 different species of bacteria. In embodiments, the composition includes less than 3 different species of bacteria. In embodiments, the composition includes less than about 2 different species of bacteria. In embodiments, the composition includes less than 2 different species of bacteria.

In embodiments, the composition is not a fecal transplant. In embodiments, the composition further includes a pharmaceutically acceptable excipient. In embodiments, the composition is a capsule, a tablet, a suspension, a suppository, a powder, a cream, an oil, an oil-in-water emulsion, a water-in-oil emulsion, or an aqueous solution. In embodiments, the composition is in the form of a powder, a solid, a semi-solid, or a liquid. In embodiments, the composition is a food or a beverage.

In embodiments, the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. is in the form of a powder. In embodiments, the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. has been lyophilized.

In embodiments, the Myxococcus sp. is in the form of spores, vegetative bacteria, or a mixture of spores and vegetative bacteria. In embodiments, the Myxococcus sp. is in the form of a powder comprising spores. In embodiments, the Clostridium sp. is in the form of spores, vegetative bacteria, or a mixture of spores and vegetative bacteria. In embodiments, the Clostridium sp. is in the form of a powder comprising spores.

In embodiments, the bacterial composition has a water activity (a_(w)) less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.9 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.9 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.8 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.8 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.7 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.7 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.6 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.6 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.5 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.5 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.4 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.4 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.3 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.3 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.2 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.2 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.1 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.1 at 20° C.

A “microbial composition” as provided herein refers to a composition including a bacterial population that comprises, consists essentially of, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp., Pediococcus sp., Bifidobacterium sp., and Clostridium sp. In embodiments, the bacterial population comprises Lactobacillus sp. and Faecalibacterium prausnitzii. In embodiments, the bacterial population comprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments, the bacterial population comprises Lactobacillus sp., and Myxococcus xanthus. In embodiments, the bacterial population comprises Lactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterial population comprises Lactobacillus sp. and Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, the bacterial population comprises Lactobacillus sp. and Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. In embodiments, the bacterial population comprises Lactobacillus sp. and Clostridium hiranonis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii. In embodiments, the bacterial population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination of the following: Lactobacillus johnsonii. Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans. Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, and Pediococcus parvulus. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In some embodiments, a microbial composition comprises one or more bacterial cells of the bacterial type Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus. In embodiments, the composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus. In embodiments, the bacteria are isolated. As used herein, a “type” or more than one “types” of bacteria may be differentiated at the genus level, the species, level, the sub-species level, the strain level or by any other taxonomic method described herein and otherwise known in the art.

In embodiments, the composition includes an effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the composition includes an effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus. In embodiments, the composition consists essentially of an effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the composition consists of an effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Where a microbial composition “consists essentially of” Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus, other agents may be included that do not interfere with the operation or basic and novel characteristics of the microbial composition.

An “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” Thus, an “effective amount” or “therapeutically effective amount” as provided herein refers to the amount of a bacterial population (e.g., a bacterial population comprising one or more species or strains of bacteria, such as a bacterial population comprising Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus) required to ameliorate or prevent the symptoms of a disease (e.g., dysbiosis, an infection, or an inflammatory disease) relative to an untreated patient. In embodiments, the microbial composition does not include Lactobacillus rhamnosus.

A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme or protein relative to the absence of the antagonist. A “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 50%, 100%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10¹⁵ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁵ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁶ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁷ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁸ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁹ to 10¹⁵ cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁸ to 10¹⁵ cfu/g. In embodiments, the composition includes 10¹² to 10¹⁵ cfu/g. In embodiments, the composition includes 10¹³ to 10¹⁵ cfu/g. In embodiments, the composition includes 10¹⁴ to 10¹⁵ cfu/g. In embodiments, the composition includes from 10³ to 10¹⁵ cfu. In embodiments, the composition includes 10⁴ to 10¹⁵ cfu. In embodiments, the composition includes 10⁵ to 10¹⁵ cfu. In embodiments, the composition includes 10⁶ to 10¹⁵ cfu. In embodiments, the composition includes 10⁷ to 10¹⁵ cfu. In embodiments, the composition includes 0l to 10¹⁵ cfu. In embodiments, the composition includes 10⁹ to 10¹⁵ cfu. In embodiments, the composition includes 10¹⁰ to 10¹⁵ cfu. In embodiments, the composition includes 10¹¹ to 10¹⁵ cfu. In embodiments, the composition includes 10¹² to 10¹⁵ cfu. In embodiments, the composition includes 10¹³ to 10¹⁵ cfu. In embodiments, the composition includes 10¹⁴ to 10¹⁵ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10¹⁴ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10¹⁴ cfu/g. In embodiments, the composition includes 10⁵ to 10¹⁴ cfu/g. In embodiments, the composition includes 10⁶ to 10¹⁴ cfu/g. In embodiments, the composition includes 10⁷ to 10¹⁴ cfu/g. In embodiments, the composition includes 10⁸ to 10¹⁴ cfu/g. In embodiments, the composition includes 10⁹ to 10¹⁴ cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹⁴ cfu/g. In embodiments, the composition includes 10¹¹ to 10¹⁴ cfu/g. In embodiments, the composition includes 10¹² to 10¹⁴ cfu/lg. In embodiments, the composition includes 10¹³ to 10¹⁴ cfu/g. In embodiments, the composition includes from 10³ to 10¹⁴ cfu. In embodiments, the composition includes 10⁴ to 10¹⁴ cfu. In embodiments, the composition includes 10⁵ to 10¹⁴ cfu. In embodiments, the composition includes 10⁶ to 10¹⁴ cfu. In embodiments, the composition includes 10⁷ to 10¹⁴ cfu. In embodiments, the composition includes 10⁸ to 10¹⁴ cfu. In embodiments, the composition includes 10⁹ to 10¹⁴ cfu. In embodiments, the composition includes 10¹⁰ to 10¹⁴ cfu. In embodiments, the composition includes 10¹¹ to 10¹⁴ cfu. In embodiments, the composition includes 10¹² to 10¹⁴ cfu. In embodiments, the composition includes 10¹³ to 10¹⁴ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10¹³ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10¹³ cfu/g. In embodiments, the composition includes 10⁵ to 10¹³ cfu/g. In embodiments, the composition includes 10⁶ to 10¹³ cfu/g. In embodiments, the composition includes 10⁷ to 10¹³ cfu/g. In embodiments, the composition includes 10⁸ to 10¹³ cfu/g. In embodiments, the composition includes 10⁹ to 10¹³ cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹³ cfu/g. In embodiments, the composition includes 10¹¹ to 10¹³ cfu/g. In embodiments, the composition includes 10¹² to 10¹³ cfu/g. In embodiments, the composition includes from 10³ to 10¹³ cfu. In embodiments, the composition includes 10⁴ to 10¹³ cfu. In embodiments, the composition includes 10⁵ to 10¹³ cfu. In embodiments, the composition includes 10⁶ to 10¹³ cfu. In embodiments, the composition includes 10⁷ to 10¹³ cfu. In embodiments, the composition includes 10⁸ to 10¹³ cfu. In embodiments, the composition includes 10⁹ to 10¹³ cfu. In embodiments, the composition includes 10¹⁰ to 10¹³ cfu. In embodiments, the composition includes 10¹¹ to 10¹³ cfu. In embodiments, the composition includes 10¹² to 10¹³ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10¹² colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10¹² cfu/g. In embodiments, the composition includes 10⁵ to 10¹² cfu/g. In embodiments, the composition includes 10⁶ to 10¹² cfu/g. In embodiments, the composition includes 10⁷ to 10¹² cfu/g. In embodiments, the composition includes 10⁸ to 10¹² cfu/g. In embodiments, the composition includes 10⁹ to 10¹² cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹² cfu/g. In embodiments, the composition includes 10¹¹ to 10¹² cfu/g. In embodiments, the composition includes from 10³ to 10¹² cfu. In embodiments, the composition includes 10⁴ to 10¹² cfu/g. In embodiments, the composition includes 10⁵ to 10¹² cfu. In embodiments, the composition includes 10⁶ to 10¹² cfu. In embodiments, the composition includes 10⁷ to 10¹² cfu. In embodiments, the composition includes 10⁸ to 10¹² cfu. In embodiments, the composition includes 10⁹ to 10¹² cfu. In embodiments, the composition includes 10¹⁰ to 10¹² cfu. In embodiments, the composition includes 10¹¹ to 10¹² cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10¹¹ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10¹¹ cfu/g. In embodiments, the composition includes 10⁵ to 10¹¹ cfu/g. In embodiments, the composition includes 10⁶ to 10¹¹ cfu/g. In embodiments, the composition includes 10⁷ to 10¹¹ cfu/g. In embodiments, the composition includes 10⁸ to 10¹¹ cfu/g. In embodiments, the composition includes 10⁹ to 10¹¹ cfu/g. In embodiments, the composition includes from 10³ to 10¹¹ cfu. In embodiments, the composition includes 10⁴ to 10¹¹ cfu. In embodiments, the composition includes 10⁵ to 10¹¹ cfu. In embodiments, the composition includes 10⁶ to 10¹¹ cfu. In embodiments, the composition includes 10⁷ to 10¹¹ cfu. In embodiments, the composition includes 10 to 10¹¹ cfu. In embodiments, the composition includes 10⁹ to 10¹¹ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10¹⁰ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10¹⁰ cfu/g. In embodiments, the composition includes 10⁵ to 10¹⁰ cfu/g. In embodiments, the composition includes 10⁶ to 10¹⁰ cfu/lg. In embodiments, the composition includes 10⁷ to 10¹⁰ cfu/g. In embodiments, the composition includes 10⁸ to 10¹⁰ cfu/g. In embodiments, the composition includes 10⁹ to 10¹⁰ cfu/g. In embodiments, the composition includes from 10³ to 10¹⁰ cfu. In embodiments, the composition includes 10⁴ to 10¹⁰ cfu. In embodiments, the composition includes 10⁵ to 10¹⁰ cfu. In embodiments, the composition includes 10⁶ to 10¹⁰ cfu. In embodiments, the composition includes 10⁷ to 10¹⁰ cfu. In embodiments, the composition includes 10⁸ to 10¹⁰ cfu. In embodiments, the composition includes 10⁹ to 10¹⁰ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10⁹ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10⁹ cfu/g. In embodiments, the composition includes 10⁵ to 10⁹ cfu/g. In embodiments, the composition includes 10⁶ to 10⁹ cfu/g. In embodiments, the composition includes 10⁷ to 10⁹ cfu/g. In embodiments, the composition includes 10⁸ to 10⁹ cfu/g. In embodiments, the composition comprises from 10³ to 10⁹ cfu. In embodiments, the composition includes 10⁴ to 10⁹ cfu. In embodiments, the composition includes 10⁵ to 10⁹ cfu. In embodiments, the composition includes 10⁶ to 10⁹ cfu. In embodiments, the composition includes 10⁷ to 10⁹ cfu. In embodiments, the composition includes 10⁸ to 10⁹ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10⁸ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10 cfu/g. In embodiments, the composition includes 10⁵ to 10⁸ cfu/g. In embodiments, the composition includes 10⁶ to 10⁸ cfu/lg. In embodiments, the composition includes 10⁷ to 10⁸ cfu/g. In embodiments, the composition includes from 10³ to 10⁸ cfu. In embodiments, the composition includes 10⁴ to 10⁸ cfu. In embodiments, the composition includes 10⁵ to 10⁸ cfu. In embodiments, the composition includes 10⁶ to 10⁸ cfu. In embodiments, the composition includes 10⁷ to 10⁸ cfu.

In embodiments, a composition provided herein may be administered orally and include live microorganisms from 10³ to 10⁷ colony forming units (cfu)/g. In embodiments, the composition includes 10⁴ to 10⁷ cfu/g. In embodiments, the composition includes 10⁵ to 10⁷ cfu/g. In embodiments, the composition includes 10⁶ to 10⁷ cfu/g. In embodiments, the composition includes from 10³ to 10⁷ cfu. In embodiments, the composition includes 10⁴ to 10⁷ cfu. In embodiments, the composition includes 10⁵ to 10⁷ cfu. In embodiments, the composition includes 10⁶ to 10⁷ cfu.

It is understood that the amount of colony forming units (cfu)/g and cfu as provided herein may refer to the amount of each bacterial species strain administered (individually) or the total cfu/g or cfu for a bacterial population.

The proportion or concentration of the compositions of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the defined microbial composition can be provided in a capsule containing from about 0.005 mg to about 1000 mg for oral administration. Alternatively or in addition, the dosage can be expressed as cfu or cfu/g of bacteria (e.g., of dry weight when expressed as cfu/g) as described above. In embodiments, the dosage may vary, but can range from the equivalent of about 10² to about 10¹⁵ cfu/g, e.g., 1×10² cfu/g, 5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g, 1×10⁴ cfu/g, 5×10⁴ cfu/g, 1×10⁵ cfu/g, 5×10⁵ cfu/g, 1×10⁶ cfu/g, 5×10⁶ cfu/g, 1×10⁷ cfu/g, 5×10⁷ cfu/g, 1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g, 5×10⁹ cfu/g, 1×10¹⁰ cfu/g, 5×10¹⁰ cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g, or 1×10¹² cfu/g of dry weight. In embodiments, Lactobacillus johnsonii. Faecalibacterium prausnitzii, Akkermansia muciniphila. Myxococcus xanthus or Pediococcus pentosaceus are administered at any one of 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10 ¹², 10¹³, 10¹⁴, or 10¹⁵ colony forming units (cfu)/g of dry weight, or total cfu, individually or total. In embodiments, the composition includes Lactobacillus johnsonii at about 10⁷ colony forming units (cfu)/g or a total of 10⁷ cfu. In embodiments, the composition includes Akkermansia muciniphila at about 10⁷ colony forming units (cfu)/g or a total of 10⁷ cfu. In embodiments, the composition includes Myxococcus xanthus at about 10⁷ colony forming units (cfu)/g or a total of 10⁷ cfu. In embodiments, the composition includes Pediococcus pentosaceus at about 10⁷ colony forming units (cfu)/g or a total of 10⁷ cfu. In embodiments, the composition includes Faecalibacterium prausnitzii at about 10 colony forming units (cfu)/g or a total of 10 cfu. In embodiments, the composition includes live microorganisms (e.g., Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus) per gram of composition, or equivalent doses calculated for inactivated or dead microorganisms or for microorganism fractions or for produced metabolites.

In embodiments, Lactobacillus johnsonii as provided herein refers to one or more isolated bacterial cells of a strain cultured from murine intestines using Lactobacillus isolation media (deMan, Rogose and Sharpe agar). Non-limiting examples of Lactobacillus johnsonii include strains deposited with ATCC under Accession Nos. 11506 and 53672.

In embodiments, Lactobacillus rhamnosus as provided herein refers to one or more isolated bacterial cells of a bacterial strain having all the identifying characteristics of a strain deposited with ATCC as Accession No. 53103; variants of the strain deposited with ATCC as Accession No. 53103 having all the identifying characteristics of the ATCC No. 53103 strain; and mutants of the strain deposited with ATCC as Accession No. 53103 having all the identifying characteristics of the ATCC No. 53103 strain.

In embodiments, Faecalibacterium prausnitzii as provided herein refers to one or more isolated bacterial cells of a bacterial strain having all the identifying characteristics of a strain deposited with ATCC as Accession No. 27766; variants of the strain deposited with ATCC as Accession No. 27766 having all the identifying characteristics of the ATCC No. 27766 strain; and mutants of the strain deposited with ATCC as Accession No. 27766 having all the identifying characteristics of the ATCC No. 27766 strain.

In embodiments, Akkermansia muciniphila as provided herein refers to one or more isolated bacterial cells of a bacterial strain having all the identifying characteristics of a strain deposited with ATCC as Accession No. BAA-835; variants of the strain deposited with ATCC as Accession No. BAA-835 having all the identifying characteristics of the ATCC No. BAA-835 strain; and mutants of the strain deposited with ATCC as Accession No. BAA-835 having all the identifying characteristics of the ATCC No. BAA-835 strain.

In embodiments, Myxococcus xanthus as provided herein refers to one or more isolated bacterial cells of a bacterial strain having all the identifying characteristics of a strain deposited with ATCC as Accession No. 25232; variants of the strain deposited with ATCC as Accession No. 25232 having all the identifying characteristics of the ATCC No. 25232 strain; and mutants of the strain deposited with ATCC as Accession No. 25232 having all the identifying characteristics of the ATCC No. 25232 strain.

In embodiments, Pediococcus pentosaceus as provided herein refers to one or more isolated bacterial cells of a bacterial strain having all the identifying characteristics of a strain deposited with ATCC as Accession No. 25744; variants of the strain deposited with ATCC as Accession No. 25744 having all the identifying characteristics of the ATCC No. 25744 strain; and mutants of the strain deposited with ATCC as Accession No. 25744 having all the identifying characteristics of the ATCC No. 25744 strain.

In embodiments, the composition is effective to increase an anti-inflammatory metabolite. In embodiments, the Lactobacillus johnsonii is effective to increase an anti-inflammatory metabolite. In embodiments, the Faecalibacterium prausnitzii is effective to increase an anti-inflammatory metabolite. In embodiments, the Akkermansia muciniphila is effective to increase an anti-inflammatory metabolite. In embodiments, the Myxococcus xanthus is effective to increase an anti-inflammatory metabolite. In embodiments, the Pediococcus pentosaceus is effective to increase an anti-inflammatory metabolite. A “metabolite” as provided herein refers to intermediates and products of the metabolism of a bacterial cell, wherein the bacterial cell resides within the gut of a mammal. The term metabolite also includes intermediates and products formed by a mammalian cell. Non-limiting examples of metabolites include amino acids, alcohols, vitamins, polyols, organic acids, nucleotides (e.g. inosine-5′-monophosphate and guanosine-5′-monophosphate), lipids, carbohydrates, peptides and proteins. An “anti-inflammatory metabolite” as provided herein refers to a metabolite produced by a cell (e.g., bacterial cell, mammalian cell) and capable of inhibiting inflammation. As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-anti-inflammatory metabolite interaction means negatively affecting (e.g., decreasing) the activity or function of the protein (e.g., decreasing the activity of an inflammatory metabolite) relative to the activity or function of the protein in the absence of the inhibitor (e.g., anti-inflammatory metabolite). The term “inhibiting” includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction, gene expression, enzymatic activity or protein expression (e.g., inflammatory metabolite) necessary for inflammation. In some embodiments inhibition refers to reduction of a disease or symptoms of disease (e.g., inflammation). Similarly an “inhibitor” is a compound (e.g., metabolite) that inhibits inflammation, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating inflammatory metabolite activity. A metabolite capable of inhibiting or decreasing inflammation as provided herein refers to a substance that results in a detectably lower activity level of inflammation of as compared to a control. The decreased activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the decrease is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or less in comparison to a control.

In embodiments, the anti-inflammatory metabolite is a microbial lipid or a microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a phospholipid. In embodiments, the anti-inflammatory metabolite is a poly-unsaturated fatty acid. In embodiments, the anti-inflammatory metabolite is microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is itoconate. In embodiments, the anti-inflammatory metabolite is n-acetylglucosamine. In embodiments, the anti-inflammatory metabolite is n-acetylgalactosamine. In embodiments, the anti-inflammatory metabolite is fucosyllactose. In embodiments, the anti-inflammatory metabolite is an amino acid. In embodiments, the anti-inflammatory metabolite is tryptophan.

In embodiments, the composition is effective to decrease a pro-inflammatory metabolite. In embodiments, the composition is effective to decrease pro-inflammatory metabolite. In embodiments, the Lactobacillus johnsonii is effective to decrease a pro-inflammatory metabolite. In embodiments, the Faecalibacterium prausnitzii is effective to decrease a pro-inflammatory metabolite. In embodiments, the Akkermansia muciniphila is effective to decrease a pro-inflammatory metabolite. In embodiments, the Myxococcus xanthus is effective to decrease pro-inflammatory metabolite. In embodiments, the Pediococcus pentosaceus is effective to decrease pro-inflammatory metabolite. A “pro-inflammatory metabolite” as provided herein refers to a metabolite produced by a cell (e.g., bacterial cell, mammalian cell) and capable of increasing inflammation. A metabolite capable of increasing inflammation as provided herein refers to a substance that results in a detectably higher level of inflammation as compared to a control. The increased activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 900/or more than that in a control. In certain instances, the increase is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid. In embodiments, the pro-inflammatory metabolite is a microbial lipid. In embodiments, the pro-inflammatory metabolite is dihydroxyoctadec-12-enoic acid, cholate or methylmalonate. In embodiments, the pro-inflammatory metabolite is a microbial carbohydrate. In embodiments, the pro-inflammatory metabolite is n-acetylymuramate, lactobionate or maltotriose. In embodiments, the pro-inflammatory metabolite is a microbial amino acid. In embodiments, the pro-inflammatory metabolite is ornithine or taurine.

The compositions provided herein may include metabolically active bacteria or metabolically inactive bacteria or fractions thereof. In embodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii, the Akkermansia muciniphila, the Myxococcus xanthus and the Pediococcus pentosaceus are metabolically active. In embodiments, the said Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus are metabolically inactive. Metabolically active bacteria are capable of dividing and produce metabolites such as carbohydrates, lipids or amino acids. In contrast metabolically inactive bacteria do not divide or produce metabolites.

III. Pharmaceutical Compositions

As described herein, the microbial compositions provided herein may include a bacterial population that comprises, consists essentially of, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp., Pediococcus sp., Bifidobacterium sp., and Clostridium sp. In embodiments, the bacterial population comprises Lactobacillus sp. and Faecalibacterium prausnitzii. In embodiments, the bacterial population comprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments, the bacterial population comprises Lactobacillus sp., and Myxococcus xanthus. In embodiments, the bacterial population comprises Lactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterial population comprises Lactobacillus sp. and Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, the bacterial population comprises Lactobacillus sp. and Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. In embodiments, the bacterial population comprises Lactobacillus sp. and Clostridium hiranonis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii. In embodiments, the bacterial population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination of the following: Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, and Pediococcus parvulus. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In some embodiments, a microbial composition comprises one or more bacterial cells of the bacterial type Lactobacillus johnsonii. Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus. In embodiments, the composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the composition includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceus. In embodiments, the bacteria are isolated bacteria.

In embodiments, the microbial composition includes a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii. Akkermansia muciniphila, Myxococcus xanthus and/or Pediococcus pentosaceus.

In embodiments, the microbial composition further includes a pharmaceutically acceptable excipient. Thus, in one aspect a pharmaceutical composition including a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and Pediococcus pentosaceus and a pharmaceutically acceptable excipient are provided.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

The microbial compositions provided herein including embodiments thereof may be adminstered orally, gastrointestinally, or rectally. Administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. In embodiments, the microbial consortium provided herein is combined with one or more excipients, for example, a disintegrant, a filler, a glidant, or a preservative. In embodiments, the microbial consortium provided herein forms part of a capsule. Suitable capsules include both hard shell capsules or soft-shelled capsules. Any lipid-based or polymer-based colloid may be used to form the capsule. Exemplary polymers useful for colloid preparations include gelatin, plant polysaccharides or their derivatives such as carrageenans and modified forms of starch and cellulose, e.g., hypromellose. Optionally, other ingredients may be added to the gelling agent solution, for example plasticizers such as glycerin and/or sorbitol to decrease the capsule's hardness, coloring agents, preservatives, disintegrants, lubricants and surface treatment.

The microbial compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.005 mg to about 2000 mg of a defined microbial consortium having minimal urease activity per dose. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.005 mg to about 1000 mg of the microbial composition provided herein.

The microbial compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mg to about 150 mg, from about 40 mg to about 100 mg, from about 50 mg to about 100 mg of Lactobacillus sp. (e.g., Lactobacillus johnsonii), Faecalibacterium sp. (Faecalibacterium prausnitzii), Akkermansia sp. (e.g., Akkermansia muciniphila), Myxococcus sp. (e.g., Myxococcus xanthus) and/or Pediococcus sp. (e.g., Pediococcus pentosaceus) individually or combined.

In some embodiments, tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

IV. Methods of Treatment

According to the methods provided herein, the subject is administered an effective amount of one or more of the agents provided herein. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response (e.g., reduction of inflammation, infection, or dysbiosis). Effective amounts and schedules for administering the agent may be determined empirically by one skilled in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)).

For prophylactic use, a therapeutically effective amount of the microbial composition described herein are administered to a subject prior to or during early onset (e.g., upon initial signs and symptoms of an autoimmune disease). Therapeutic treatment involves administering to a subject a therapeutically effective amount of the agents described herein after diagnosis or development of disease. Thus, in another aspect, a method of treating a disease (e.g., an inflammatory disease, an infection, or dysbiosis) in a subject in need thereof is provided.

The terms “subject,” “patient,” “individual,” etc. are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice.

As used herein, “treating” or “treatment of” a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 200, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition (e.g., inflammation, infection, or dysbiosis). For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50°, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

Compositions comprising a defined microbial compositions can be administered to the gastrointestinal tract of a subject by nasoduodenal catheter, by enema, or by endoscopy, enteroscopy, or colonoscopy or orally in a consumable capsule or pill. In certain embodiments, the defined microbial compositions are diluted in a suitable excipient (e.g., saline solution). In a preferred embodiment, the bacteria are delivered in lyophilized form.

Regardless of how the compositions are formulated, the dosage required will depend on the route of administration, the nature of the formulation, the nature of the subject's condition, e.g., immaturity of the immune system or a gastrointestinal disorder, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians. In embodiments, suitable dosages are in the range of 0.01-1,000 mg/kg. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. In embodiments, the dose can be, for example, 1 mg/kg, 2 mg/kg, 5 mg kg, 10 mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg. Alternatively or in addition, the dosage can be expressed as cfu or as cfu/g of dry weight. In embodiments, the dosage may vary, but can range from the equivalent of about 10² to about 10¹² cfu/g, e.g., 1×10² cfu/g, 5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g, 1×10⁴ cfu/g, 5×10⁴ cfu/g, 1×10⁵ cfu/g, 5×10⁵ cfu/g, 1×10⁶ cfu/g, 5×10⁶ cfu/g, 1×10⁷ cfu/g, 5×10⁷ cfu/g, 1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g, 5×10⁹ cfu/g, 1×10¹⁰ cfu/g, 5×10¹⁰ cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g, or 1×10¹² cfu/g of dry weight of any one of the administered bacteria (individually) or of the total population of bacteria. In embodiments, the dosage can range from about 10² to about 10¹² cfu, e.g., 1×10² cfu, 5×10² cfu, 1×10³ cfu, 5×10³ cfu, 1×10⁴ cfu, 5×10⁴ cfu, 1×10⁵ cfu, 5×10⁵ cfu, 1×10⁶ cfu, 5×10⁶ cfu, 1×10⁷ cfu, 5×10⁷ cfu, 1×10⁸ cfu, 5×10⁸ cfu, 1×10⁹ cfu, 5×10⁹ cfu, 1×10¹⁰ cfu, 5×10¹⁰ cfu, 1×10¹¹ cfu, 5×10¹¹ cfu, or 1×10¹² cfu of any one of the administered bacteria (individually) or of the total population of bacteria.

Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, a composition can be administered 1, 2, 3, 4, 5, 6, or 7 times a week (for, for example, 4 weeks to many months or years); once a month (for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compositions can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.

The compositions may also be administered in conjunction with other therapeutic agents. Other therapeutic agents will vary according to the particular disorder, but can include, for example, dietary modification, hemodialysis, therapeutic agents such as sodium benzoate, phenylacetate, arginine, or surgical remedies. Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

Provided herein are methods of treating and preventing inflammatory diseases, infections (such as respiratory or gastrointestinal infections) and dysbiosis comprising administering the bacterial populations or microbial compositions described herein including embodiments thereof.

In an aspect, a method of treating or preventing dysbiosis, an inflammatory disease, or a viral respiratory infection, in a subject in need thereof is provided.

In an aspect, a method of increasing the level of an anti-inflammatory compound and/or decreasing the level of a pro-inflammatory compound in a subject in need thereof is provided.

In an aspect, a method of altering the metabolism of a subject in need thereof is provided.

In embodiments, the method includes administering to the subject an effective amount of a bacterial population that comprises, consists essentially of, or consists of, 1, 2, 3, 4, 5, 6, 7, or 8 (or at least 1, 2, 3, 4, 5, 6, 7, or 8) bacterial species. In embodiments, the bacterial population comprises, consists essentially of, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp., Pediococcus sp., Bifidobacterium sp., and Clostridium sp. In embodiments, the bacterial population comprises Lactobacillus sp. and Faecalibacterium prausnitzii. In embodiments, the bacterial population comprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments, the bacterial population comprises Lactobacillus sp., and Myxococcus xanthus. In embodiments, the bacterial population comprises Lactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterial population comprises Lactobacillus sp. and Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, the bacterial population comprises Lactobacillus sp. and Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. In embodiments, the bacterial population comprises Lactobacillus sp. and Clostridium hiranonis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. In embodiments, the Lactobacillus sp. is Lactobacillus johnsonii. In embodiments, the bacterial population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination of the following: Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, and Pediococcus parvulus. In embodiments, the bacterial population includes Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In embodiments, the bacteria are isolated bacteria.

In embodiments, the method includes administering to the subject an effective amount of a bacterial population including Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp. In embodiments, (i) the Lactobacillus sp. is Lactobacillus johnsonii; (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. is Pediococcus pentosaceus. In embodiments, (i) the Lactobacillus sp. is Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis; (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. is Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus.

In embodiments, the Myxococcus sp. is in the form of spores, vegetative bacteria, or a mixture of spores and vegetative bacteria. In embodiments, the Myxococcus sp. is in the form of a powder comprising spores. In embodiments, the Clostridium sp. is in the form of spores, vegetative bacteria, or a mixture of spores and vegetative bacteria. In embodiments, the Clostridium sp. is in the form of a powder comprising spores.

In embodiments, less than about 20, 15, 10, 9, 8, 7, or 6 different species of bacteria are administered to the subject. In embodiments, less than about 20 different species of bacteria are administered to the subject. In embodiments, less than 20 different species of bacteria are administered to the subject. In embodiments, less than about 15 different species of bacteria are administered to the subject. In embodiments, less than 15 different species of bacteria are administered to the subject. In embodiments, less than about 10 different species of bacteria are administered to the subject. In embodiments, less than 10 different species of bacteria are administered to the subject. In embodiments, less than about 9 different species of bacteria are administered to the subject. In embodiments, less than 9 different species of bacteria are administered to the subject. In embodiments, less than about 8 different species of bacteria are administered to the subject. In embodiments, less than 8 different species of bacteria are administered to the subject. In embodiments, less than about 7 different species of bacteria are administered to the subject. In embodiments, less than 7 different species of bacteria are administered to the subject. In embodiments, less than about 6 different species of bacteria are administered to the subject. In embodiments, less than 6 different species of bacteria are administered to the subject.

In embodiments, the bacterial population forms part of a bacterial composition. In embodiments, the bacterial composition includes less than about 20, 15, 10, 9, 8, 7, or 6 species of bacteria. In embodiments, the bacterial composition includes less than about 20 species of bacteria. In embodiments, the bacterial composition includes less than 20 species of bacteria. In embodiments, the bacterial composition includes less than about 15 species of bacteria. In embodiments, the bacterial composition includes less than 15 species of bacteria. In embodiments, the bacterial composition includes less than about 10 species of bacteria. In embodiments, the bacterial composition includes less than 10 species of bacteria. In embodiments, the bacterial composition includes less than about 9 species of bacteria. In embodiments, the bacterial composition includes less than 9 species of bacteria. In embodiments, the bacterial composition includes less than about 8 species of bacteria. In embodiments, the bacterial composition includes less than 8 species of bacteria. In embodiments, the bacterial composition includes less than about 7 species of bacteria. In embodiments, the bacterial composition includes less than 7 species of bacteria. In embodiments, the bacterial composition includes less than about 6 species of bacteria. In embodiments, the bacterial composition includes less than 6 species of bacteria.

In embodiments, the bacterial composition further includes a pharmaceutically acceptable excipient. In embodiments, the bacterial composition is not a fecal transplant. In embodiments, the bacterial composition is a capsule, a tablet, a suspension, a suppository, a powder, a cream, an oil, an oil-in-water emulsion, a water-in-oil emulsion, or an aqueous solution. In embodiments, the bacterial composition is in the form of a powder, a solid, a semi-solid, or a liquid.

In embodiments, the bacterial composition has a water activity (a_(w)) less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.9 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.9 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.8 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.8 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.7 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.7 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.6 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.6 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.5 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.5 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.4 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.4 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.3 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.3 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.2 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.2 at 20° C. In embodiments, the bacterial composition has an a_(w) less than about 0.1 at 20° C. In embodiments, the bacterial composition has an a_(w) less than 0.1 at 20° C.

In embodiments, the bacterial composition is a food or a beverage.

In embodiments, the bacterial composition is administered orally or rectally.

In embodiments, the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. is in the form of a powder. In embodiments, the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. has been lyophilized.

In embodiments, the subject is a human. In embodiments, the subject suffers from or resides with someone who suffers from a bacterial, viral, or fungal gastrointestinal infection.

In embodiments, the subject has an inflammatory disease. In embodiments, the subject is at risk of suffering from an inflammatory disease. In embodiments, the subject has at least 1, 2, 3, or 4 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease. In embodiments, the subject has at least 4 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease. In embodiments, the subject has at least 3 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease. In embodiments, the subject has at least 2 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease. In embodiments, the subject has at least 1 cousin, grandparent, parent, aunt, uncle, and/or sibling who has been diagnosed with an inflammatory disease.

In embodiments, the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

In embodiments, the inflammatory disease is pediatric allergic asthma or inflammatory bowel disease. In embodiments, the subject suffers from constipation, diarrhea, bloating, urgency, and/or abdominal pain.

In embodiments, the subject has been administered an antibiotic within the last 1, 2, 3, or 4 months. In embodiments, the subject has been administered an antibiotic within the last 4 months.

In embodiments, the subject has been administered an antibiotic within the last 3 months. In embodiments, the subject has been administered an antibiotic within the last 2 months. In embodiments, the subject has been administered an antibiotic within the last 1 month.

In embodiments, the subject is a neonate. In embodiments, the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old. In embodiments, the subject is less than about 1 month old. In embodiments, the subject is less than 1 month old. In embodiments, the subject is less than about 2 months old. In embodiments, the subject is less than 2 months old. In embodiments, the subject is less than about 3 months old. In embodiments, the subject is less than 3 months old. In embodiments, the subject is less than about 4 months old. In embodiments, the subject is less than 4 months old. In embodiments, the subject is less than about 5 months old. In embodiments, the subject is less than 5 months old. In embodiments, the subject is less than about 6 months old. In embodiments, the subject is less than 6 months old. In embodiments, the subject is less than about 7 months old. In embodiments, the subject is less than 7 months old. In embodiments, the subject is less than about 8 months old. In embodiments, the subject is less than 8 months old. In embodiments, the subject is less than about 9 months old. In embodiments, the subject is less than 9 months old. In embodiments, the subject is less than about 12 months old. In embodiments, the subject is less than 12 months old. In embodiments, the subject is less than about 18 months old. In embodiments, the subject is less than 18 months old. In embodiments, the subject is less than about 24 months old. In embodiments, the subject is less than 24 months old.

In embodiments, the subject is between about 2 and about 18 years old, or is at least about 18 years old. In embodiments, the subject is between 2 and 18 years old, or is at least 18 years old. In embodiments, the subject is between about 2 and about 18 years old, or is at least about 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 19 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 19 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 20 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 20 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 25 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 25 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 30 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 30 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 40 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 40 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 50 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 50 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 60 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 60 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 70 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 70 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 80 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 80 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 90 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 90 years old.

In embodiments, the subject comprises a gastrointestinal microbiome that (a) has an increased proportion of Streptococcus spp., Bifidobacterium spp., and Enterococcus spp. compared to a healthy or general population; (b) has a reduced proportion of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae compared to a healthy or general population; (c) has an increased proportion of Candida albicans and Debaryomyces spp. compared to a healthy or general population; (d) has a reduced proportion of Bifdobacteria spp., Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp. compared to a healthy or general population; (e) has a reduced proportion of Malassezia spp. compared to a healthy or general population; (f) has an increased proportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., or Bifidobacterium spp. compared to a healthy or general population; or (g) has an increased proportion of Enterococcus faecalis, Enterococcus faecium, or Clostridium difficile compared to a healthy or general population.

In embodiments, the effective amount is effective to (i) increase the level of a Bifidobacterium sp., Clostridia sp. belonging to Clade IV or XIV, a Lachnospira sp., and/or a Ruminococcus sp. in the subject; (ii) lower the pH in the feces of the subject; (iii) increase the level of lactic acid in the feces of the subject; (iv) increase the level of circulating itaconate in the subject; (v) treat, reduce, or prevent allergic inflammation in a subject; (vi) reduce an adaptive immune response in an airway of the subject; (vii) reduce dendritic cell activation in a gastrointestinal-associated mesenteric lymoph node; (viii) increase the level of repair macrophages in the lungs, blood, serum, or plasma of the subject; (ix) increase the level of an anti-inflammatory compound in the subject; (x) decrease the level of a pro-inflammatory compound in the subject; (xi) decrease the level of eotaxin expression and/or secretion in the subject; and/or (xii) decrease the level of mucin expression and/or secretion in the subject.

In embodiments, the effective amount is effective to decrease the level of mucin secretion and/or secretion in the lungs of the subject.

In embodiments, the anti-inflammatory compound is a cytokine, a microbial lipid, a microbial carbohydrate, or a microbial amino acid. In embodiments, the anti-inflammatory compound is IL-17. In embodiments,

In embodiments, the pro-inflammatory compound is a cytokine, a microbial lipid, a microbial carbohydrate, or a microbial amino acid. In embodiments, the pro-inflammatory compound is IL-4, IL-10, IL-8, IL-13, TNF-α, or MUC5B.

In embodiments, the Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. is metabolically active. In embodiments, the Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. is metabolically inactive.

In embodiments, the method further includes administering (a) a Bifidobacterium sp., (b) Cystobacter sp., or (c) a fungal microorganism to the subject.

In embodiments, the effective amount is effective to alter the metabolism of the subject. In embodiments, altering the metabolism of the subject includes increasing the level of a lipid, a phospholipid, or a plasmalogen. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 2 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 3 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 4 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 5 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 6 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 7 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 8 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 9 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 10 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 11 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 12 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 13 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 14 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 15 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 16 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 17 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 18 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 19 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 20 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 21 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 22 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 23 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 24 compounds listed in Table 3, in the subject. In embodiments, altering the metabolism of the subject includes increasing the level of any compound listed in Table 3, or any combination of 25 compounds listed in Table 3, in the subject. In embodiments, the level is increased in the feces of the subject. In embodiments, the level is increased in a body fluid of the subject. In embodiments, altering the metabolism of the subject comprises decreasing the level of a carbohydrate, a lipid, or an energy compound in a subject. In embodiments, altering the metabolism of the subject comprises decreasing the level of any compound listed in Table 4, or any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 2 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 3 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 4 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 5 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 6 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 7 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 8 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 9 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 10 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 11 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 12 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 13 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 14 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 15 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 16 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 17 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 18 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 19 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 20 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 21 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 22 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 23 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 24 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 25 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 30 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 35 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 40 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 45 compounds listed in Table 4, in the subject. In embodiments, altering the metabolism of the subject includes decreasing the level of any compound listed in Table 4, or any combination of 50 compounds listed in Table 4, in the subject. In embodiments, the level is decreased in the feces of the subject. In embodiments, the level is decreased in a body fluid of the subject.

In an aspect is provided a method of treating or preventing an inflammatory disease in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of Lactobacillus johnsonii. Faecalibacterium prausnitzii. Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus form a microbial composition as provided herein. Where the Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus form a microbial composition, the bacteria form part of a composition including a pharmaceutically acceptable carrier for administration to and colonialization of the gut. Acceptable carriers include, but are not limited to inulin. In embodiments, the gut is of a healthy subject. In embodiments, the gut is of a subject in need of treatment or prevention of an inflammatory disease. In embodiments, the subject is a neonate. A “neonate” as provided herein refers to a newborn child or mammal. In embodiments, the neonate is less than about four weeks old.

In an aspect, a method of treating or preventing an inflammatory disease in a subject in need thereof is provided. The method including administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

In embodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus are metabolically active. “Metabolically active” as provided herein refer to cells (e.g., bacteria) capable of cell division. In embodiments the metabolically active cell is capable of substrate (e.g. glucose) consumption. In embodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus are metabolically inactive. In embodiments, the microbial composition is effective for administration to the gut. In embodiments, the microbial composition does not include Lactobacillus rhamnosus.

In embodiments, the microbial composition is effective to increase an anti-inflammatory metabolite (e.g., microbial lipid, a microbial carbohydrate or a microbial amino acid). As described herein, the anti-inflammatory metabolite may be a microbial lipid (e.g., phospholipid, poly-unsaturated fatty acid). In embodiments, the anti-inflammatory metabolite is a phospholipid. In embodiments, the anti-inflammatory metabolite is poly-unsaturated fatty acid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate (e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine, fucosyllactose). In embodiments, the anti-inflammatory metabolite is itoconate. In embodiments, the anti-inflammatory metabolite is n-acetylglucosamine. In embodiments, the anti-inflammatory metabolite is n-acetylgalactosamine. In embodiments, the anti-inflammatory metabolite is fucosyllactose. In embodiments, the anti-inflammatory metabolite is a microbial amino acid (e.g., tryptophan). In embodiments, the anti-inflammatory metabolite is tryptophan. A composition capable of increasing an anti-inflammatory metabolite as provided herein refers to a composition that results in a detectably higher level of an anti-inflammatory metabolite as compared to a control. The increased activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that in a control. In certain instances, the increase is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control. In embodiments, the microbial composition is effective to increase the number of IL-17-secreting T helper cells.

In embodiments, the microbial composition is effective to decrease a pro-inflammatory metabolite. As described herein, the pro-inflammatory metabolite may be a microbial lipid (e.g., dihydroxyoctadec-12-enoic acid, cholate or methylmalonate). In embodiments, the pro-inflammatory metabolite is dihydroxyoctadec-12-enoic acid. In embodiments, the pro-inflammatory metabolite is a cholate. In embodiments, the pro-inflammatory metabolite is methylmalonate. In embodiments, the pro-inflammatory metabolite is a microbial carbohydrate (e.g., n-acetylymuramate, lactobionate or maltotriose). In embodiments, the pro-inflammatory metabolite is n-acetylymuramate. In embodiments, the pro-inflammatory metabolite is lactobionate. In embodiments, the pro-inflammatory metabolite is maltotriose. In embodiments, the pro-inflammatory metabolite is a microbial amino acid (e.g., ornithine or taurine). In embodiments, the pro-inflammatory metabolite is ornithine. In embodiments, the pro-inflammatory metabolite is taurine. A composition capable of decreasing a pro-inflammatory metabolite as provided herein refers to a composition that results in a detectably lower level of a pro-inflammatory metabolite as compared to a control. The decreased activity can be 10%, 20%, 300, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the decrease is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or less in comparison to a control.

In embodiments, the pro-inflammatory metabolite is IL-4, IL-10, IL-13 or MUC5B. In embodiments, the pro-inflammatory metabolite is IL-4. In embodiments, the pro-inflammatory metabolite is IL-10. In embodiments, the pro-inflammatory metabolite is IL-13. In embodiments, the pro-inflammatory metabolite MUC5B. In embodiments, the pro-inflammatory metabolite MUC5AC. In embodiments, the microbial composition is effective to decrease T helper cell type 2 cytokine expression.

The term “IL-4” as provided herein includes any of the recombinant or naturally-occurring forms of the interleukin 4 (IL-4) cytokine or variants or homologs thereof that maintain IL-4 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL-4). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-4 polypeptide. In embodiments, 1-4 is the protein as identified by the NCBI sequence reference GI:4504669 (Accession No. NP_000580.1; SEQ ID NO: 1), or an isoform, a homolog or functional fragment thereof.

The term “IL-10” as provided herein includes any of the recombinant or naturally-occurring forms of the interleukin 10 (IL-10) cytokine or variants or homologs thereof that maintain IL-10 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL-10). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99⁰% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-10 polypeptide. In embodiments, IL-10 is the protein as identified by the NCBI sequence reference GI:10835141 (Accession No. NP_000563.1; SEQ ID NO:2), or an isoform, a homolog or functional fragment thereof.

The term “IL-13” as provided herein includes any of the recombinant or naturally-occurring forms of the interleukin 13 (IL-13) cytokine or variants or homologs thereof that maintain IL-13 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96° %, 97%, 98%, 99% or 100% activity compared to IL-13). In some aspects, the variants or homologs have at least 90° %, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-13 polypeptide. In embodiments, IL-13 is the protein as identified by the NCBI sequence reference GI:26787978 (Accession No. NP_002179.2; SEQ ID NO:3), or an isoform, a homolog or functional fragment thereof.

The term “IL-17” as provided herein includes any of the recombinant or naturally-occurring forms of the interleukin 17 (IL-17) cytokine or variants or homologs thereof that maintain IL-17 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL-17). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-17 polypeptide. In embodiments, IL-17 is the protein as identified by the UniProt sequence reference Q16552 (SEQ ID NO:4), or a homolog or functional fragment thereof. In embodiments, IL-17 is the protein as identified by the UniProt sequence reference Q9UHF5, or an isoform, a homolog or functional fragment thereof.

The term “MUC5AC” as provided herein includes any of the recombinant or naturally-occurring forms of the mucin 5AC (MUC5AC) protein or variants or homologs thereof that maintain MUC5AC protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to MUC5AC). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%0, amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MUC5AC polypeptide. In embodiments, MUC5AC is the protein as identified by the UniProt sequence reference P98088 (SEQ ID NO:5), or an isoform, a homolog or functional fragment thereof.

The term “MUC5B” as provided herein includes any of the recombinant or naturally-occurring forms of the mucin 5B (MUC5B) protein or variants or homologs thereof that maintain MUC5B protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%0/activity compared to MUC5B). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MUC5B polypeptide. In embodiments, MUC5B is the protein as identified by the UniProt sequence reference Q9HC84 (SEQ ID NO:6), or an isoform, a homolog or functional fragment thereof.

In embodiments, the method further includes administering a therapeutically effective amount of a fungus. In embodiments, the fungus is a Malassezia fungus. In embodiments, the microbial composition is effective to decrease a pathogenic fungal activity. A “pathogenic fungal activity” as referred to herein is a metabolic activity derived from a pathogenic fungus. In embodiments, the pathogenic fungus is Candida albicans. In embodiments, the pathogenic fungus forms a pro-inflammatory lipid.

In embodiments, the subject is a neonate. In embodiments, the neonate is less than about four weeks old. In embodiments, the neonate is treated for at least about one month. In embodiments, the neonate is treated for at least about two months. In embodiments, the neonate is treated for at least about three months. In embodiments, the neonate is treated for at least about four months. In embodiments, the neonate is treated for at least about five months. In embodiments, the neonate is treated for at least about six months.

In embodiments, the neonate is treated for about one month. In embodiments, the neonate is treated for about two months. In embodiments, the neonate is treated for about three months. In embodiments, the neonate is treated for about four months. In embodiments, the neonate is treated for about five months. In embodiments, the neonate is treated for about six months.

In embodiments, the neonate is treated for less than about one month. In embodiments, the neonate is treated for less than about two months. In embodiments, the neonate is treated for less than about three months. In embodiments, the neonate is treated for less than about four months. In embodiments, the neonate is treated for less than about five months. In embodiments, the neonate is treated for less than about six months.

In embodiments, the neonate is treated for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, weeks.

In embodiments, the inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum or erythema nodosum. In embodiments, the inflammatory disease is asthma. In embodiments, the inflammatory disease is ulcerative colitis. In embodiments, the inflammatory disease is irritable bowel syndrome. In embodiments, the inflammatory disease is arthritis. In embodiments, the inflammatory disease is uveitis. In embodiments, the inflammatory disease is pyoderma gangrenosum. In embodiments, the inflammatory disease is erythema nodosum.

In an aspect, a method of increasing an anti-inflammatory metabolite in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii. Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the method further includes a pharmaceutically active excipient as provided herein. The Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus may form a microbial composition provided herein including embodiments thereof. Thus, in embodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus form a microbial composition.

The anti-inflammatory metabolite may be a microbial lipid (e.g., a phospholipid, a poly unsaturated fatty acid), a microbial carbohydrate (e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine, fucosyllactose) or a microbial amino acid (e.g., tryptophan) as provided herein. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is a microbial amino acid.

In an aspect is provided a method of decreasing a pro-inflammatory metabolite in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, the method further includes a pharmaceutically active excipient as provided herein. The Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus may form a microbial composition provided herein including embodiments thereof. Thus, in embodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus form a microbial composition.

As described herein, the pro-inflammatory metabolite may be a microbial lipid (e.g., dihydroxyoctadec-12-enoic acid, cholate or methylmalonate). In embodiments, the pro-inflammatory metabolite is dihydroxyoctadec-12-enoic acid. In embodiments, the pro-inflammatory metabolite is a cholate. In embodiments, the pro-inflammatory metabolite is methylmalonate. In embodiments, the pro-inflammatory metabolite is a microbial carbohydrate (e.g., n-acetylymuramate, lactobionate or maltotriose). In embodiments, the pro-inflammatory metabolite is n-acetylymuramate. In embodiments, the pro-inflammatory metabolite is lactobionate. In embodiments, the pro-inflammatory metabolite is maltotriose. In embodiments, the pro-inflammatory metabolite is a microbial amino acid (e.g., ornithine or taurine). In embodiments, the pro-inflammatory metabolite is ornithine. In embodiments, the pro-inflammatory metabolite is taurine.

In an aspect, a method of increasing the level of an anti-inflammatory compound and/or decreasing the level of a pro-inflammatory compound in a subject in need thereof is provided. In embodiments, the method includes administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, the method for increasing the level of the anti-inflammatory compound increases and/or decreases the level of the pro-inflammatory compound in the feces, blood, plasma, serum, broncheoalveolar lavage fluid, sweat, saliva, sputum, lymph, spinal fluid, urine, tears, bile, aqueous humour, vitreous humour, aminiotic fluid, breast milk, cerebrospinal fluid, cerumen, nasal mucus, phlegm, or sebum of the subject.

In embodiments, the anti-inflammatory compound is a microbial lipid, a microbial carbohydrate, or a microbial amino acid.

In embodiments, the subject suffers from dysbiosis or an inflammatory disease.

In embodiments, the inflammatory disease is a disease as described herein. In embodiments, the inflammatory disease is ulcerative colitis. In embodiments, the inflammatory disease is irritable bowel syndrome. In embodiments, the inflammatory disease is arthritis. In embodiments, the inflammatory disease is uveitis. In embodiments, the inflammatory disease is pyoderma gangrenosum. In embodiments, the inflammatory disease is erythema nodosum.

In an aspect, a method of treating or preventing a viral respiratory infection in a subject in need thereof is provided. The method including administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, wherein the viral respiratory infection is caused by a respiratory syncytial virus, an influenza virus, a parainfluenza virus, an adenovirus, a coronavirus, or a rhinovirus. In embodiments, the viral respiratory infection is bronchiolitis, a cold, croup, or pneumonia.

In aspects is provided a method of treating or preventing an allergy in a subject in need thereof. The method including administering to the subject an effective amount of a bacteria population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, the allergy is an allergy to milk, eggs, fish, shellfish, a tree nut, peanuts, wheat, dander from a cat, dog, or rodent, an insect sting, pollen, latex, dust mites, or soybeans. In embodiments, the allergy is pediatric allergic asthma, hay fever, or allergic airway sensitization.

V. Methods of Detection

In an aspect a method of detecting an anti-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease is provided. The method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of an anti-inflammatory metabolite in the biological sample. The anti-inflammatory metabolite may be a microbial lipid (e.g., a phospholipid, a poly unsaturated fatty acid), a microbial carbohydrate (e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine, fucosyllactose) or a microbial amino acid (e.g., tryptophan) as provided herein. Thus, in embodiments, the anti-inflammatory metabolite is a microbial lipid or a microbial carbohydrate as described herein. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate.

In embodiments, the expression level of a compound (e.g., a metabolite) is the amount (e.g., weight) of the compound. In embodiments, the expression level of a compound (e.g., a protein such as a cytokine) is the level of mRNA expression.

In an aspect a method of detecting a pro-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease is provided. The method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of a pro-inflammatory metabolite in the biological sample. The pro-inflammatory metabolite may be a microbial lipid (e.g., dihydroxyoctadec-12-enoic acid, cholate or methylmalonate), a microbial carbohydrate (e.g., n-acetylymuramate, lactobionate or maltotriose syllactose) or a microbial amino acid (e.g., ornithine or taurine) as provided herein.

In an aspect, a method of detecting a pro-inflammatory compound in a subject in need thereof is provided. In embodiments, the method includes (i) obtaining a biological sample from the subject; and (ii) detecting the pro-inflammatory compound in the biological sample.

In an aspect, a method of monitoring the effect of treatment for dysbiosis or an inflammatory disease is provided. In embodiments, the method includes (i) obtaining a biological sample from the subject; and (ii) detecting whether the biological sample is pro-inflammatory.

In an aspect, a method of determining an inflammatory disease activity in a subject is provided. In embodiments, the method includes (i) obtaining a biological sample from the subject; and (ii) detecting whether the biological sample is pro-inflammatory.

In an aspect, a method of detecting an anti-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease is provided. In embodiments, the method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of an anti-inflammatory metabolite in the biological sample.

In embodiments, the subject has or is at risk for developing dysbiosis. In embodiments, the subject has an inflammatory disease. In embodiments, the subject is at risk of suffering from an inflammatory disease.

In embodiments, the subject (i) has at least 1, 2, 3, or 4 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease; (ii) suffers from constipation, diarrhea, bloating, urgency, and/or abdominal pain; and/or (iii) has been administered an antibiotic within the last 1, 2, or 4 months.

In embodiments, the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

In embodiments, the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old. In embodiments, the subject is less than about 1 month old. In embodiments, the subject is less than 1 month old. In embodiments, the subject is less than about 2 months old. In embodiments, the subject is less than 2 months old. In embodiments, the subject is less than about 3 months old. In embodiments, the subject is less than 3 months old. In embodiments, the subject is less than about 4 months old. In embodiments, the subject is less than 4 months old. In embodiments, the subject is less than about 5 months old. In embodiments, the subject is less than 5 months old. In embodiments, the subject is less than about 6 months old. In embodiments, the subject is less than 6 months old. In embodiments, the subject is less than about 7 months old. In embodiments, the subject is less than 7 months old. In embodiments, the subject is less than about 8 months old. In embodiments, the subject is less than 8 months old. In embodiments, the subject is less than about 9 months old. In embodiments, the subject is less than 9 months old. In embodiments, the subject is less than about 12 months old. In embodiments, the subject is less than 12 months old. In embodiments, the subject is less than about 18 months old. In embodiments, the subject is less than 18 months old. In embodiments, the subject is less than about 24 months old. In embodiments, the subject is less than 24 months old.

In embodiments, the subject is between about 2 and about 18 years old, or is at least about 18 years old. In embodiments, the subject is between 2 and 18 years old, or is at least 18 years old. In embodiments, the subject is between about 2 and about 18 years old, or is at least about 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 19 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 19 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 20 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 20 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 25 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 25 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 30 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 30 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 40 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 40 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 50 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 50 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 60 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 60 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 70 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 70 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 80 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 80 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 90 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 90 years old.

In embodiments, the subject comprises a gastrointestinal microbiome that (a) has an increased proportion of Streptococcus spp., Bifidobacterium spp., and Enterococcus spp. compared to a healthy or general population; (b) has a reduced proportion of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae compared to a healthy or general population; (c) has an increased proportion of Candida albicans and Debaryomyces spp. compared to a healthy or general population; (d) has a reduced proportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp. compared to a healthy or general population; (e) has a reduced proportion of Malassezia spp. compared to a healthy or general population; (f) has an increased proportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., or Bifidobacterium spp. compared to a healthy or general population; or (g) has an increased proportion of Enterococcus faecalis, Enterococcus faecium, or Clostridium difficile compared to a healthy or general population.

In embodiments, the biological sample is a bodily fluid. In embodiments, wherein the bodily fluid is blood, plasma, serum, fecal water, or a brancheoaleolar lavage. In embodiments, the bodily fluid is fecal water.

In embodiments, detecting the pro-inflammatory compound includes contacting an antigen presenting cell with the biological sample. In embodiments, the antigen presenting cell is a dendritic cell. In embodiments, the dendritic cell has been isolated from blood. In embodiments, the dendritic cell has been isolated from the blood of a healthy subject (e.g., a subject who does not have an inflammatory disease, an infection, and who has not been administered an antibiotic within about 1, 2, 3, 4, 5, or 6 months). In embodiments, the dendritic cell has been obtained (e.g., isolated, selected, or enriched) from peripheral blood mononuclear cells. In embodiments, the dendritic cell is part of a primary culture of dendritic cells. In embodiments, the dendritic cell is part of a culture of dendritic cells that has been passaged less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In embodiments, the dendritic cell is not immortalized. In embodiments, the dendritic cell is an immortalized dendritic cell.

In embodiments, detecting the pro-inflammatory compound further includes contacting a naïve T cell with the antigen presenting cell to produce a contacted T cell. In embodiments, the method further includes detecting a cytokine produced by the contacted T cell and/or the progeny of the contacted T cell. In embodiments, the T cell has been isolated from blood. In embodiments, the T cell has been isolated from the blood of a healthy subject (e.g., a subject who does not have an inflammatory disease, an infection, and who has not been administered an antibiotic within about 1, 2, 3, 4, 5, or 6 months). In embodiments, the T cell has been obtained (e.g., isolated, selected, or enriched) from peripheral blood mononuclear cells. In embodiments, the T cell is part of a primary culture of T cells. In embodiments, the T cell is part of a culture of T cells that has been passaged less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In embodiments, the T cell is not immortalized. In embodiments, the T cell is an immortalized T cell.

In embodiments, the pro-inflammatory compound is detected if (i) the proportion of T-helper (TH)-2 cells is increased in the progeny of the contacted T cell compared to a control; (ii) the proportion of TH-1, TH-17, and/or TH22 cells is increased in the progeny of the contacted T cell compared to a control; (iii) the ratio of TH-1 cells to TH-2 cells is decreased in the progeny of the contacted T cell compared to a control; (iv) the proportion of IL-17 producing CD8+ T cells is increased in the progeny of the contacted T cell compared to a control; and/or (v) the amount of IL-4, IL-10, and/or IL-13 produced by the progeny of the contacted T cell and/or the progeny thereof is increased compared to a control.

In embodiments, the control is (i) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with sterile culture medium and/or the progeny thereof; (ii) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with an antigen presenting cell that has been contacted with a biological sample from a subject who does not have dysbiosis, an inflammatory disease, or a gastrointestinal infection, and/or the progeny thereof; and/or (iii) a reference value corresponding to the proportion, ratio, and/or amount in the general population or a population of subjects who do not have dysbiosis, an inflammatory disease, or a gastrointestinal infection.

In embodiments, the method further includes directing the subject to receive treatment or further testing or monitoring for dysbiosis or an inflammatory disease if the pro-inflammatory compound is detected in the subject.

In embodiments, the method further includes administering the composition as described herein including embodiments thereof to the subject if the pro-inflammatory compound is detected in the subject.

In embodiments, the method further includes diagnosing the subject as having or at risk of developing dysbiosis or an inflammatory disease if the pro-inflammatory compound is detected in the subject.

In embodiments, a method of determining whether a subject has or is at risk of developing dysbiosis or an inflammatory disease is provided. In embodiments, the method includes (i) obtaining a biological sample from the subject; and (ii) detecting a pro-inflammatory compound according to a method described herein including embodiments thereof.

In some examples of the disclosed methods, when the expression level of an anti-inflammatory metabolite or pro-inflammatory metabolite is assessed, the level is compared with a control expression level of the anti-inflammatory metabolite or pro-inflammatory metabolite. By control level is meant the expression level of a particular an anti-inflammatory metabolite or pro-inflammatory metabolite from a sample or subject lacking a disease (e.g. an inflammatory disease), at a selected stage of a disease or disease state, or in the absence of a particular variable such as a therapeutic agent. Alternatively, the control level comprises a known amount of anti-inflammatory metabolite or pro-inflammatory metabolite. Such a known amount correlates with an average level of subjects lacking a disease, at a selected stage of a disease or disease state, or in the absence of a particular variable such as a therapeutic agent. A control level also includes the expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites from one or more selected samples or subjects as described herein. For example, a control level includes an assessment of the expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a sample from a subject that does not have a disease (e.g. an inflammatory disease), is at a selected stage of progression of a disease (e.g. inflammatory disease), or has not received treatment for a disease. Another exemplary control level includes an assessment of the expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolite in samples taken from multiple subjects that do not have a disease, are at a selected stage of progression of a disease, or have not received treatment for a disease.

When the control level includes the expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a sample or subject in the absence of a therapeutic agent (e.g., the microbial composition provided herein including embodiments thereof), the control sample or subject is optionally the same sample or subject to be tested before or after treatment with a therapeutic agent or is a selected sample or subject in the absence of the therapeutic agent. Alternatively, a control level is an average expression level calculated from a number of subjects without a particular disease. A control level also includes a known control level or value known in the art.

In embodiments, the biological sample is a bodily fluid. In embodiments, the bodily fluid is serum, fecal water or brancheoaleolar lavage. In embodiments, the bodily fluid is serum. In embodiments, the bodily fluid is fecal water. In embodiments, the bodily fluid is brancheoaleolar lavage. In embodiments, the biological sample is a tissue. In embodiments, the tissue is lung, spleen, or ileum tissue. In embodiments, the biological sample is a cell. In embodiments, the biological sample is a lung cell. In embodiments, the biological sample is a spleen cell. In embodiments, the biological sample is an ileum cell. In embodiments, the sample includes one or more bacterial cells.

In embodiments, a biological sample is a bodily fluid obtained by filtration and/or centrifugation. For example, the biological sample may be a filtrate of e.g., blood or feces or the supernatant of centrifuged blood or feces. In embodiments, a filtrate is centrifuged. In embodiments a supernatant is filtered. In embodiments, centrifugation is used to increase the passage of a fluid through a filter. Non-limiting examples of filters include filters that restrict any molecule greater than, e.g., 50, 100, 200, 300, 400, 500, 50-500, 50-100, 100-500 nm in diameter (or average diameter), or greater than 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 25, 50, 100, or 200 microns in diameter (e.g., average diameter). In embodiments, a filter has pores of about 50, 100, 200, 300, 400, 500, 50-500, 50-100, 100-500 nm in diameter or about 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 25, 50, 100, or 200 microns in diameter.

In embodiments, detecting a compound (e.g., a metabolite) and/or the expression level thereof comprises High performance liquid chromatography (HPLC), gas chromatography, liquid chromatography, Mass spectrometry (MS), inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS) and spark source mass spectrometry (SSMS), matrix-assisted laser desorption/ionization (MALDI), and/or MALDI-TOF.

In embodiments, detecting the expression level of a compound comprises lysing a cell. In embodiments, detecting the expression level of a compound comprises a polymerase chain reaction (e.g., reverse transcriptase polymerase chain reaction), microarray analysis, immunohistochemistry, or flow cytometry.

In embodiments, the determining includes: (a) contacting in vitro the anti-inflammatory metabolite with an antigen presenting cell, thereby forming a metabolite-antigen presenting cell; (b) contacting the metabolite-antigen presenting cell with a T cell, thereby forming a contacted T cell; and (c) detecting a cytokine produced by the contacted T cell. In embodiments, the cytokine is produced by an activated or differentiating T cell.

In embodiments, the determining includes: (a) contacting in vitro the pro-inflammatory metabolite with an antigen presenting cell, thereby forming a metabolite-antigen presenting cell; (b) contacting the metabolite-antigen presenting cell with a T cell, thereby forming a contacted T cell; (c) detecting a cytokine produced by the contacted T cell.

In embodiments, the inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum. In embodiments, the inflammatory disease is asthma. In embodiments, the inflammatory disease is ulcerative colitis. In embodiments, the inflammatory disease is irritable bowel syndrome. In embodiments, the inflammatory disease is arthritis. In embodiments, the inflammatory disease is uveitis. In embodiments, the inflammatory disease is pyoderma gangrenosum. In embodiments, the inflammatory disease is erythema nodosum.

In an aspect, a method of determining whether a subject has or is at risk of developing an inflammatory disease is provided. The method includes (i) detecting an expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an pro-inflammatory metabolite or a decreased expression level of an anti-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In an aspect, a method of determining whether a subject has or is at risk of developing an inflammatory disease is provided. The method includes (i) detecting an expression level of one or more pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein an increased expression level of an pro-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In another aspect, a method of determining whether a subject has or is at risk of developing an inflammatory disease is provided. The method includes (i) detecting an expression level of one or more anti-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein a decreased expression level of an anti-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In embodiments, the anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid as provided herein. The anti-inflammatory metabolite may be a microbial lipid (e.g., a phospholipid, a poly unsaturated fatty acid), a microbial carbohydrate (e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine, fucosyllactose) or a microbial amino acid (e.g., tryptophan) as provided herein. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is a microbial amino acid.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid as provided herein. The pro-inflammatory metabolite may be a microbial lipid (e.g., dihydroxyoctadec-12-enoic acid, cholate or methylmalonate), a microbial carbohydrate (e.g., n-acetylymuramate, lactobionate or maltotriose syllactose) or a microbial amino acid (e.g., ornithine or taurine) as provided herein.

In embodiments, the inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum. In embodiments, the inflammatory disease is asthma. In embodiments, the inflammatory disease is ulcerative colitis. In embodiments, the inflammatory disease is irritable bowel syndrome. In embodiments, the inflammatory disease is arthritis. In embodiments, the inflammatory disease is uveitis. In embodiments, the inflammatory disease is pyoderma gangrenosum. In embodiments, the inflammatory disease is erythema nodosum.

In an aspect, a method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy is provided. The method includes (i) determining a first expression level of an anti-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of an anti-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of an anti-inflammatory metabolite to the first expression level of an anti-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject.

In embodiments, the anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid as provided herein. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is a microbial amino acid.

In an aspect, a method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy is provided. The method includes (i) determining a first expression level of a pro-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of a pro-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of a pro-inflammatory metabolite to the first expression level of a pro-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid as provided herein. In embodiments, the pro-inflammatory metabolite is a microbial lipid. In embodiments, the pro-inflammatory metabolite is a microbial carbohydrate. In embodiments, the pro-inflammatory metabolite is a microbial amino acid.

In embodiments, the inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum. In embodiments, the inflammatory disease is asthma. In embodiments, the inflammatory disease is ulcerative colitis. In embodiments, the inflammatory disease is irritable bowel syndrome. In embodiments, the inflammatory disease is arthritis. In embodiments, the inflammatory disease is uveitis. In embodiments, the inflammatory disease is pyoderma gangrenosum. In embodiments, the inflammatory disease is erythema nodosum.

In an aspect, a method of determining whether a subject has or is at risk of developing dysbiosis or an inflammatory disease is provided. The method including: (i) obtaining a biological sample from the subject; and (ii) detecting whether the biological sample is pro-inflammatory.

In embodiments, the subject suffers from or resides with someone who suffers from a bacterial, viral, or fungal gastrointestinal infection.

In embodiments, the subject (i) has at least 1, 2, 3, or 4 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease; (ii) suffers from constipation, diarrhea, bloating, urgency, and/or abdominal pain; and/or (iii) has been administered an antibiotic within the last 1, 2, or 4 months.

In embodiments, the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

In embodiments, the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old. In embodiments, the subject is less than about 1 month old. In embodiments, the subject is less than 1 month old. In embodiments, the subject is less than about 2 months old. In embodiments, the subject is less than 2 months old. In embodiments, the subject is less than about 3 months old. In embodiments, the subject is less than 3 months old. In embodiments, the subject is less than about 4 months old. In embodiments, the subject is less than 4 months old. In embodiments, the subject is less than about 5 months old. In embodiments, the subject is less than 5 months old. In embodiments, the subject is less than about 6 months old. In embodiments, the subject is less than 6 months old. In embodiments, the subject is less than about 7 months old. In embodiments, the subject is less than 7 months old. In embodiments, the subject is less than about 8 months old. In embodiments, the subject is less than 8 months old. In embodiments, the subject is less than about 9 months old. In embodiments, the subject is less than 9 months old. In embodiments, the subject is less than about 12 months old. In embodiments, the subject is less than 12 months old. In embodiments, the subject is less than about 18 months old. In embodiments, the subject is less than 18 months old. In embodiments, the subject is less than about 24 months old. In embodiments, the subject is less than 24 months old.

In embodiments, the subject is between about 2 and about 18 years old, or is at least about 18 years old. In embodiments, the subject is between 2 and 18 years old, or is at least 18 years old. In embodiments, the subject is between about 2 and about 18 years old, or is at least about 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 19 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 19 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 20 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 20 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 25 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 25 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 30 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 30 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 40 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 40 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 50 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 50 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 60 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 60 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 70 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 70 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 80 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 80 years old. In embodiments, the subject is between about 2 and about 18 years old, or is about 90 years old. In embodiments, the subject is between about 2 and about 18 years old, or is 90 years old.

In embodiments, the subject comprises a gastrointestinal microbiome that (a) has an increased proportion of Streptococcus spp., Bifidobacterium spp., and Enterococcus spp. compared to a healthy or general population; (b) has a reduced proportion of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae compared to a healthy or general population; (c) has an increased proportion of Candida albicans and Debaryomyces spp. compared to a healthy or general population; (d) has a reduced proportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp. compared to a healthy or general population; (e) has a reduced proportion of Malassezia spp. compared to a healthy or general population; (f) has an increased proportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., or Bifidobacterium spp. compared to a healthy or general population; or (g) has an increased proportion of Enterococcus faecalis, Enterococcus faecium, or Clostridium difficile compared to a healthy or general population.

In embodiments, the biological sample is a bodily fluid. In embodiments, the bodily fluid is blood, plasma, serum, fecal water, or a brancheoaleolar lavage. In embodiments, the bodily fluid is fecal water.

In embodiments, detecting whether the biological sample is pro-inflammatory includes contacting an antigen presenting cell with the biological sample. In embodiments, the antigen presenting cell is a dendritic cell.

In embodiments, detecting whether the biological sample is pro-inflammatory further includes contacting a naïve T cell with the antigen presenting cell to produce a contacted T cell.

In embodiments, the method further includes detecting a cytokine produced by the contacted T cell and/or the progeny of the contacted T cell.

In embodiments, the biological sample is detected to be pro-inflammatory if (i) the proportion of T-helper (TH)-2 cells is increased in the progeny of the contacted T cell compared to a control; (ii) the proportion of TH-1, TH-17, and/or TH22 cells is increased in the progeny of the contacted T cell compared to a control; (iii) the ratio of TH-1 cells to TH-2 cells is decreased in the progeny of the contacted T cell compared to a control; (iv) the proportion of IL-17 producing CD8+ T cells is increased in the progeny of the contacted T cell compared to a control; and/or (v) the amount of IL-4, IL-10, and/or IL-13 produced by the progeny of the contacted T cell and/or the progeny thereof is increased compared to a control.

In embodiments, the control is (i) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with sterile culture medium and/or the progeny thereof; (ii) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with an antigen presenting cell that has been contacted with a biological sample from a subject who does not have dysbiosis, an inflammatory disease, or a gastrointestinal infection, and/or the progeny thereof, and/or (iii) a reference value corresponding to the proportion, ratio, and/or amount in the general population or a population of subjects who do not have dysbiosis, an inflammatory disease, or a gastrointestinal infection.

In embodiments, the method further includes directing the subject to receive treatment or further testing or monitoring for dysbiosis or an inflammatory disease if the biological sample is detected to be pro-inflammatory.

In embodiments, the method further includes administering a bacterial population or composition as described herein including embodiments thereof to the subject if the biological sample is detected to be pro-inflammatory.

In embodiments, the subject includes a gastrointestinal microbiome that (a) has an increased proportion of Streptococcus spp., Bifidobacterium spp., and Enterococcus spp. compared to a healthy or general population; (b) has a reduced proportion of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae compared to a healthy or general population; (c) has an increased proportion of Candida albicans and Debaryomyces spp. compared to a healthy or general population; (d) has a reduced proportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp. compared to a healthy or general population; (e) has a reduced proportion of Malassezia spp. compared to a healthy or general population; (f) has an increased proportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., or Bifidobacterium spp. compared to a healthy or general population; or (g) has an increased proportion of Enterococcus faecalis, Enterococcus faecium, or Clostridium difficile compared to a healthy or general population.

In embodiments, the method further includes determining whether the subject has a gastrointestinal microbiome that (a) has an increased proportion of Streptococcus spp., Bifidobacterium spp., and Enterococcus spp. compared to a healthy or general population; (b) has a reduced proportion of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae compared to a healthy or general population; (c) has an increased proportion of Candida albicans and Debaryomyces spp. compared to a healthy or general population; (d) has a reduced proportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp. compared to a healthy or general population; (e) has a reduced proportion of Malassezia spp. compared to a healthy or general population; (f) has an increased proportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., or Bifidobacterium spp. compared to a healthy or general population; or (g) has an increased proportion of Enterococcus faecalis, Enterococcus faecium, or Clostridium difficile compared to a healthy or general population.

In embodiments, a method of treating or preventing dysbiosis, a viral respiratory infection, or an inflammatory disease in a subject determined to have or be at risk of developing dysbiosis, a viral respiratory infection, or an inflammatory disease according to a method described herein including embodiments thereof. In embodiments, the method includes administering a bacterial population disclosed herein to the subject.

In another aspect, a method of determining an inflammatory disease activity in a subject is provided. The method includes (i) detecting an expression level of one or more anti-inflammatory metabolites in a subject; (ii) determining whether the expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in the subject; and (iii) based at least in part on the expression level in step (ii), determining the inflammatory disease activity in the subject.

In embodiments, the anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid as provided herein. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is a microbial amino acid.

In another aspect, a method of determining an inflammatory disease activity in a subject is provided. The method includes (i) detecting an expression level of one or more pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in the subject; and (iii) based at least in part on the expression level in step (ii), determining the inflammatory disease activity in the subject.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid as provided herein. In embodiments, the anti-inflammatory metabolite is a microbial lipid. In embodiments, the anti-inflammatory metabolite is a microbial carbohydrate. In embodiments, the anti-inflammatory metabolite is a microbial amino acid.

In embodiments, the inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum. In embodiments, the inflammatory disease is asthma. In embodiments, the inflammatory disease is ulcerative colitis. In embodiments, the inflammatory disease is irritable bowel syndrome. In embodiments, the inflammatory disease is arthritis. In embodiments, the inflammatory disease is uveitis. In embodiments, the inflammatory disease is pyoderma gangrenosum. In embodiments, the inflammatory disease is erythema nodosum.

In an aspect, a method of determining whether a subject has or is at risk of developing dysbiosis or an inflammatory disease is provided. The method including: (i) detecting an expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an pro-inflammatory metabolite or a decreased expression level of an anti-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease, and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

In an aspect, a method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy including: (i) determining a first expression level of an anti-inflammatory or pro-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of an anti-inflammatory or pro-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of an anti-inflammatory or pro-inflammatory metabolite to the first expression level of an anti-inflammatory or pro-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject is provided.

In an aspect, a method of determining an inflammatory disease activity in a subject is provided. The method including: (i) detecting an expression level of one or more anti-inflammatory or pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in the subject; and (iii) based at least in part on the expression level in step (ii), determining the inflammatory disease activity in the subject.

TABLE 1 Non-limiting examples of Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp., and Pediococcus sp. that can be used singly, or in any combination in bacterial populations of methods and compositions provided herein. Phylum Class Order Family Genus Species Verruco- Verruco- Verruco- Verruco- Akkermansia Akkermansia microbia microbiae microbiales microbiaceae muciniphila Firmicutes Clostridia Clostridiales Rumino- Faecali- Faecali- coccaceae bacterium bacterium prausnitzii Proteo- Deltaproteo- Myxococcales unclassified sfA unclassified bacteria bacteria Proteo- Deltaproteo- Myxococcales Cystobacteraceae Cystobacter Cystobacter bacteria bacteria fuscus Proteo- Deltaproteo- Myxococcales Myxococcaceae Myxococcus Myxococcus bacteria bacteria xanthus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus zeae (Lacto bacillus rhamnosus) Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus acidipiscis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus acidophilus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus agilis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus aviarius Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus brevis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus coleohominis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus crispatus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus crustorum Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus curvatus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus diolivorans Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus farraginis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus fermentum Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus fuchuensis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus harbinensis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus helveticus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus hilgardii Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus intestinalis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus jensenii Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus johnsonii Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus kefiranofaciens Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus kefiri Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus lindneri Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus mali Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus manihotivorans Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus mucosae Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus oeni Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus oligofermentans Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus panis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus pantheris Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus parabrevis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus paracollinoides Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus parakefiri Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus paraplantarum Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus pentosus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus pontis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus reuteri Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus rossiae Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus salivarius Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus siliginis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus sucicola Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus vaccinostercus Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus vaginalis Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillus vini Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactococcus garvieae Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Lactococcus lactis Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus Pediococcus pentosaceus Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus Pediococcus acidilactici Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus Pediococcus damnosus Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus Pediococcus ethanolidurans Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus Pediococcus parvulus Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium Bifidobacterium bifidum Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium Bifidobacterium pseudolongum Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium Bifidobacterium saeculare Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium Bifidobacterium subtile Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium Clostridium hiranonis

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EMBODIMENTS

Embodiments include P1 to P34 following.

Embodiment P1

A method of treating or preventing an inflammatory disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus.

Embodiment P2

The method of embodiment 1, further comprising a pharmaceutically active excipient.

Embodiment P3

The method of embodiment 1 or 2, wherein said Lactobacillus johnsonii. Faecalibacterium prausnitzii, Akkermansia muciniphila. Myxococcus xanthus and Pediococcus pentosaceus form a microbial composition.

Embodiment P4

The method of embodiment 3, wherein said microbial composition is effective for administration to the gut.

Embodiment P5

The method of embodiment 3, wherein said microbial composition is effective to increase an anti-inflammatory metabolite.

Embodiment P6

The method of embodiment 3, wherein said microbial composition is effective to decrease a pro-inflammatory metabolite.

Embodiment P7

The method of embodiment 5, wherein said anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P8

The method of embodiment 6, wherein said pro-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P9

The method of embodiment 8, wherein said pro-inflammatory metabolite is IL-4. IL-10, IL-13 or MUC5B.

Embodiment P10

The method of one of embodiments 1 or 9, wherein said Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus are metabolically active.

Embodiment P11

The method of one of embodiment 1 or 9, wherein said Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus are metabolically inactive.

Embodiment P12

The method of one of embodiments 1-11, further comprising administering a therapeutically effective amount of a fungus.

Embodiment P13

The method of one of embodiments 1-12, wherein said subject is a neonate.

Embodiment P14

The method of one of embodiments 1-13, wherein said inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P15

A method of increasing an anti-inflammatory metabolite in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus.

Embodiment P16

The method of embodiment 15, further comprising a pharmaceutically active excipient.

Embodiment P17

The method of embodiment 15 or 16, wherein said Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus form a microbial composition.

Embodiment P18

The method of one of embodiments 15-17, wherein said anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P19

The method of one of embodiments 15-18, wherein said inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P20

A method of detecting an anti-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease, said method comprising: (i) obtaining a biological sample from said subject; and (ii) determining an expression level of an anti-inflammatory metabolite in said biological sample.

Embodiment P21

The method of embodiment 20, wherein said anti-inflammatory metabolite is a microbial lipid or a microbial carbohydrate.

Embodiment P22

The method of embodiment 20 or 21, wherein said biological sample is a bodily fluid.

Embodiment P23

The method of embodiment 22, wherein said bodily fluid is serum, fecal water or brancheoaleolar lavage.

Embodiment P24

The method of one of embodiments 20-23, wherein said determining comprises: (a) contacting in vitro said anti-inflammatory metabolite with an antigen presenting cell, thereby forming a metabolite-antigen presenting cell; (b) contacting said metabolite-antigen presenting cell with a T cell, thereby forming a contacted T cell; and (c) detecting a cytokine produced by said contacted T cell.

Embodiment P25

The method of one of embodiments 20-24, wherein said inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P26

A method of determining whether a subject has or is at risk of developing an inflammatory disease, said method comprising: (i) detecting an expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a subject; (ii) determining whether said expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an pro-inflammatory metabolite or a decreased expression level of an anti-inflammatory metabolite relative to said standard control indicates that said subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on said expression level in step (ii), determining whether said subject has or is at risk for developing an inflammatory disease.

Embodiment P27

The method of embodiment 26, wherein said inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P28

The method of embodiment 26, wherein said anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P29

A method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy comprising: (i) determining a first expression level of an anti-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of an anti-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of an anti-inflammatory metabolite to the first expression level of an anti-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject.

Embodiment P30

The method of embodiment 29, wherein said inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P31

The method of embodiment 29, wherein said anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P32

A method of determining an inflammatory disease activity in a subject, said method comprising: (i) detecting an expression level of one or more anti-inflammatory metabolites in a subject; (ii) determining whether said expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in said subject; and (iii) based at least in part on said expression level in step (ii), determining said inflammatory disease activity in said subject.

Embodiment P33

The method of embodiment 32, wherein said inflammatory disease is asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P34

The method of embodiment 32, wherein said anti-inflammatory metabolite is a microbial lipid, a microbial carbohydrate or a microbial amino acid.

Further embodiments include embodiments 1 to 111 following.

Embodiment 1

A method of treating or preventing dysbiosis in a subject in need thereof, the method comprising administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 2

The method of embodiment 1, wherein (i) the Lactobacillus sp. is Lactobacillus johnsonii; (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. is Pediococcus pentosaceus.

Embodiment 3

The method of embodiment 1 or 2, wherein (i) the Lactobacillus sp. is Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mall, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis; (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. is Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, or Pediococcus parvulus.

Embodiment 4

The method of any one of embodiments 1 to 3, wherein the Myxococcus sp. is in the form of spores, vegetative bacteria, or a mixture of spores and vegetative bacteria.

Embodiment 5

The method of embodiment 4, wherein the Myxococcus sp. is in the form of a powder comprising spores.

Embodiment 6

The method of any one of embodiments 1 to 5, wherein less than about 20, 15, 10, 9, 8, 7, or 6 different species of bacteria are administered to the subject.

Embodiment 7

The method of embodiment 1, wherein the bacterial population forms part of a bacterial composition.

Embodiment 8

The method of embodiment 7, wherein the bacterial composition comprises less than about 20, 15, 10, 9, 8, 7, or 6 species of bacteria.

Embodiment 9

The method of embodiment 7 or 8, wherein the bacterial composition is not a fecal transplant.

Embodiment 10

The method of any one of embodiments 7 to 9, wherein the bacterial composition further comprises a pharmaceutically acceptable excipient.

Embodiment 11

The method of any one of embodiments 7 to 10, wherein the bacterial composition is a capsule, a tablet, a suspension, a suppository, a powder, a cream, an oil, an oil-in-water emulsion, a water-in-oil emulsion, or an aqueous solution.

Embodiment 12

The method of any one of embodiments 7 to 10, wherein the bacterial composition is in the form of a powder, a solid, a semi-solid, or a liquid.

Embodiment 13

The method of any one of embodiments 7 to 12, wherein the bacterial composition has a water activity (a_(w)) less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C.

Embodiment 14

The method of any one of embodiments 7 to 13, wherein the bacterial composition is a food or a beverage.

Embodiment 15

The method of any one of embodiments 7 to 14, wherein the bacterial composition is administered orally or rectally.

Embodiment 16

The method of any one of embodiments 1 to 15, wherein the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. is in the form of a powder.

Embodiment 17

The method of any one of embodiments 1 to 16, wherein the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. has been lyophilized.

Embodiment 18

The method of any one of embodiments 1 to 17, wherein the subject is a human.

Embodiment 19

The method of any one of embodiments 1 to 18, wherein the subject suffers from or resides with someone who suffers from a bacterial, viral, or fungal gastrointestinal infection.

Embodiment 20

The method of any one of embodiments 1 to 19, wherein the subject has an inflammatory disease.

Embodiment 21

The method of any one of embodiments 1 to 20, wherein the subject is at risk of suffering from an inflammatory disease.

Embodiment 22

The method of any one of embodiments 1 to 21, wherein the subject has at least 1, 2, 3, or 4 cousins, grandparents, parents, aunts, uncles, and/or siblings who have been diagnosed with an inflammatory disease.

Embodiment 23

The method of any one of embodiments 20 to 22, wherein the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

Embodiment 24

The method of embodiment 23, wherein the inflammatory disease is pediatric allergic asthma or inflammatory bowel disease.

Embodiment 25

The method of any one of embodiments 1 to 24, wherein the subject suffers from constipation, diarrhea, bloating, urgency, and/or abdominal pain.

Embodiment 26

The method of any one of embodiments 1 to 25, wherein the subject has been administered an antibiotic within the last 1, 2, 3, or 4 months.

Embodiment 27

The method of any one of embodiments 1 to 26, wherein the subject is a neonate.

Embodiment 28

The method of any one of embodiments 1 to 26, wherein the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old.

Embodiment 29

The method of any one of embodiments 1 to 26, wherein the subject is between about 2 and about 18 years old, or is at least about 18 years old.

Embodiment 30

The method of any one of embodiments 1 to 29, wherein the subject comprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,         Bifidobacterium spp., and Enterococcus spp. compared to a         healthy or general population;     -   (b) has a reduced proportion of Alternaria alternata,         Aspergillus flavus, Aspergillus cibarius, and Candida sojae         compared to a healthy or general population;     -   (c) has an increased proportion of Candida albicans and         Debaryomyces spp. compared to a healthy or general population;     -   (d) has a reduced proportion of Bifidobacteria spp.,         Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.         compared to a healthy or general population;     -   (e) has a reduced proportion of Malassezia spp. compared to a         healthy or general population;     -   (f) has an increased proportion of Bacterioides spp.,         Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.         compared to a healthy or general population; or     -   (g) has an increased proportion of Enterococcus faecalis,         Enterococcus faecium, or Clostridium difficile compared to a         healthy or general population.

Embodiment 31

The method of any one of embodiments 1 to 30, wherein the effective amount is effective to

-   -   (i) increase the level of a Bifidobacterium sp., Clostridia sp.         belonging to Clade IV or XIV, a Lachnospira sp., and/or a         Ruminococcus sp. in the subject;     -   (ii) lower the pH in the feces of the subject;     -   (iii) increase the level of lactic acid in the feces of the         subject;     -   (iv) increase the level of circulating itaconate in the subject;     -   (v) treat, reduce, or prevent allergic inflammation in a         subject;     -   (vi) reduce an adaptive immune response in an airway of the         subject;     -   (vii) reduce dendritic cell activation in a         gastrointestinal-associated mesenteric lymoph node;     -   (viii) increase the level of repair macrophages in the lungs,         blood, serum, or plasma of the subject;     -   (ix) increase the level of an anti-inflammatory compound in the         subject;     -   (x) decrease the level of a pro-inflammatory compound in the         subject;     -   (xi) decrease the level of eotaxin expression and/or secretion         in the subject; and/or decrease the level of mucin expression         and/or secretion in the subject.

Embodiment 32

The method of embodiment 31, wherein the effective amount is effective to decrease the level of mucin secretion and/or secretion in the lungs of the subject.

Embodiment 33

The method of embodiment 31 or 32, wherein the anti-inflammatory compound is a cytokine, a microbial lipid, a microbial carbohydrate, or a microbial amino acid.

Embodiment 34

The method of embodiment 33, wherein the anti-inflammatory compound is IL-17.

Embodiment 35

The method of any one of embodiments 31 to 34, wherein the pro-inflammatory compound is a cytokine, a microbial lipid, a microbial carbohydrate, or a microbial amino acid.

Embodiment 36

The method of embodiment 35, wherein the pro-inflammatory compound is IL-4, IL-10, IL-8, IL-13, TNF-α, or MUC5B.

Embodiment 37

The method of one of embodiments 1 or 36, wherein the Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. is metabolically active.

Embodiment 38

The method of one of embodiments 1 or 36, wherein the Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. is metabolically inactive.

Embodiment 39

The method of any one of embodiments 1 to 38, further comprising administering (a) a Bifidobacterium sp., (b) Cystobacter sp., or (c) a fungal microorganism to the subject.

Embodiment 40

A method of treating or preventing an inflammatory disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 41

The method of embodiment 40, wherein the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

Embodiment 42

A method of treating or preventing a viral respiratory infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 43

The method of embodiment 42, wherein the viral respiratory infection is caused by a respiratory syncytial virus, an influenza virus, a parainfluenza virus, an adenovirus, a coronavirus, or a rhinovirus.

Embodiment 44

The method of embodiment 42 or 43, wherein the viral respiratory infection is bronchiolitis, a cold, croup, or pneumonia.

Embodiment 45

A method of treating or preventing an allergy in a subject in need thereof, the method comprising administering to the subject an effective amount of a bacteria population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 46

The method of embodiment 45, wherein the allergy is an allergy to milk, eggs, fish, shellfish, a tree nut, peanuts, wheat, dander from a cat, dog, or rodent, an insect sting, pollen, latex, dust mites, or soybeans.

Embodiment 47

The method of embodiment 45 or 46, wherein the allergy is pediatric allergic asthma, hay fever, or allergic airway sensitization.

Embodiment 48

A method of increasing the level of an anti-inflammatory compound and/or decreasing the level of a pro-inflammatory compound in a subject in need thereof, comprising administering to the subject an effective amount of a bacterial population comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 49

The method of embodiment 48, for increasing the level of the anti-inflammatory compound increases and/or decreases the level of the pro-inflammatory compound in the feces, blood, plasma, serum, broncheoalveolar lavage fluid, sweat, saliva, sputum, lymph, spinal fluid, urine, tears, bile, aqueous humour, vitreous humour, aminiotic fluid, breast milk, cerebrospinal fluid, cerumen, nasal mucus, phlegm, or sebum of the subject.

Embodiment 50

The method of one of embodiments 48 or 49, wherein the anti-inflammatory compound is a microbial lipid, a microbial carbohydrate, or a microbial amino acid.

Embodiment 51

The method of any one of embodiments 48 to 50, wherein subject suffers from dysbiosis or an inflammatory disease.

Embodiment 52

A composition comprising Lactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 53

The composition of embodiment 52, wherein (i) the Lactobacillus sp. is Lactobacillus johnsonii, (ii) the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. is Pediococcus pentosaceus.

Embodiment 54

The composition of embodiment 52 or 53, wherein the composition comprises less than about 20, 15, 10, 9, 8, 7, or 6 different species of bacteria.

Embodiment 55

The composition of any one of embodiments 52 to 54, wherein the composition is not a fecal transplant.

Embodiment 56

The composition of any one of embodiments 52 to 55, further comprising a pharmaceutically acceptable excipient.

Embodiment 57

The composition of any one of embodiments 52 to 56, which is a capsule, a tablet, a suspension, a suppository, a powder, a cream, an oil, an oil-in-water emulsion, a water-in-oil emulsion, or an aqueous solution.

Embodiment 58

The composition of any one of embodiments 52 to 57, which is in the form of a powder, a solid, a semi-solid, or a liquid.

Embodiment 59

The composition of any one of embodiments 52 to 58, which has a water activity (a_(w)) less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C.

Embodiment 60

The composition of any one of embodiments 52 to 59, which is a food or a beverage.

Embodiment 61

The composition of any one of embodiments 52 to 60, wherein the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. is in the form of a powder.

Embodiment 62

The composition of any one of embodiments 52 to 61, wherein the Lactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. has been lyophilized.

Embodiment 63

A method of detecting a pro-inflammatory compound in a subject in need thereof, comprising: (i) obtaining a biological sample from the subject; and (ii) detecting the pro-inflammatory compound in the biological sample.

Embodiment 64

The method of embodiment 63, wherein the subject has or is at risk for developing dysbiosis.

Embodiment 65

The method of embodiments 63 or 64, wherein the subject has an inflammatory disease.

Embodiment 66

The method of any one of embodiments 63 to 65, wherein the subject is at risk of suffering from an inflammatory disease.

Embodiment 67

The method of any one of embodiments 63 to 66, wherein the subject

-   -   (i) has at least 1, 2, 3, or 4 cousins, grandparents, parents,         aunts, uncles, and/or siblings who have been diagnosed with an         inflammatory disease;     -   (ii) suffers from constipation, diarrhea, bloating, urgency,         and/or abdominal pain; and/or     -   (iii) has been administered an antibiotic within the last 1, 2,         or 4 months.

Embodiment 68

The method any one of embodiments 63 to 67, wherein the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

Embodiment 69

The method of any one of embodiments 63 to 69, wherein the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old.

Embodiment 70

The method of any one of embodiments 63 to 69, wherein the subject is between about 2 and about 18 years old, or is at least about 18 years old.

Embodiment 71

The method of any one of embodiments 63 to 70, wherein the subject comprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,         Bifidobacterium spp., and Enterococcus spp. compared to a         healthy or general population;     -   (b) has a reduced proportion of Alternaria alternata,         Aspergillus flavus, Aspergillus cibarius, and Candida sojae         compared to a healthy or general population;     -   (c) has an increased proportion of Candida albicans and         Debaryomyces spp. compared to a healthy or general population;     -   (d) has a reduced proportion of Bifidobacteria spp.,         Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.         compared to a healthy or general population;     -   (e) has a reduced proportion of Malassezia spp. compared to a         healthy or general population     -   (f) has an increased proportion of Bacterioides spp.,         Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.         compared to a healthy or general population; or     -   (g) has an increased proportion of Enterococcus faecalis,         Enterococcus faecium, or Clostridium difficile compared to a         healthy or general population.

Embodiment 72

The method of any one of embodiments 63 to 71, wherein the biological sample is a bodily fluid.

Embodiment 73

The method of embodiment 72, wherein the bodily fluid is blood, plasma, serum, fecal water, or a brancheoaleolar lavage.

Embodiment 74

The method of embodiment 72 or 73, wherein the bodily fluid is fecal water.

Embodiment 75

The method of any one of embodiments 63 to 74, wherein detecting the pro-inflammatory compound comprises contacting an antigen presenting cell with the biological sample.

Embodiment 76

The method of embodiment 75, wherein the antigen presenting cell is a dendritic cell.

Embodiment 77

The method of any one of embodiments 63 to 74, wherein detecting the pro-inflammatory compound further comprises contacting a naïve T cell with the antigen presenting cell to produce a contacted T cell.

Embodiment 78

The method of embodiment 77, further comprising detecting a cytokine produced by the contacted T cell and/or the progeny of the contacted T cell.

Embodiment 79

The method of any one of embodiments 77 or 78, wherein the pro-inflammatory compound is detected if

-   -   (i) the proportion of T-helper (TH)-2 cells is increased in the         progeny of the contacted T cell compared to a control;     -   (ii) the proportion of TH-1, TH-17, and/or TH22 cells is         increased in the progeny of the contacted T cell compared to a         control;     -   (iii) the ratio of TH-1 cells to TH-2 cells is decreased in the         progeny of the contacted T cell compared to a control;     -   (iv) the proportion of IL-17 producing CD8+ T cells is increased         in the progeny of the contacted T cell compared to a control;         and/or     -   (v) the amount of IL-4, IL-10, and/or IL-13 produced by the         progeny of the contacted T cell and/or the progeny thereof is         increased compared to a control.

Embodiment 80

The method of embodiment 79, wherein the control is (i) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with sterile culture medium and/or the progeny thereof; (ii) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with an antigen presenting cell that has been contacted with a biological sample from a subject who does not have dysbiosis, an inflammatory disease, or a gastrointestinal infection, and/or the progeny thereof; and/or (iii) a reference value corresponding to the proportion, ratio, and/or amount in the general population or a population of subjects who do not have dysbiosis, an inflammatory disease, or a gastrointestinal infection.

Embodiment 81

The method of any one of embodiments 63 to 80, further comprising directing the subject to receive treatment or further testing or monitoring for dysbiosis or an inflammatory disease if the pro-inflammatory compound is detected in the subject.

Embodiment 82

The method of any one of embodiments 63 to 81, further comprising administering the composition of any one of embodiments 52 to 62 to the subject if the pro-inflammatory compound is detected in the subject.

Embodiment 83

The method of any one of embodiments 63 to 82, further comprising diagnosing the subject as having or at risk of developing dysbiosis or an inflammatory disease if the pro-inflammatory compound is detected in the subject.

Embodiment 84

A method of determining whether a subject has or is at risk of developing dysbiosis or an inflammatory disease, the method comprising: (i) obtaining a biological sample from the subject; and (ii) detecting a pro-inflammatory compound according to the method of any one of embodiments 63 to 80.

Embodiment 85

A method of determining whether a subject has or is at risk of developing dysbiosis or an inflammatory disease, the method comprising: (i) obtaining a biological sample from the subject; and (ii) detecting whether the biological sample is pro-inflammatory.

Embodiment 86

The method of embodiment 85, wherein the subject suffers from or resides with someone who suffers from a bacterial, viral, or fungal gastrointestinal infection.

Embodiment 87

The method of embodiment 85 or 86, wherein the subject

-   -   (i) has at least 1, 2, 3, or 4 cousins, grandparents, parents,         aunts, uncles, and/or siblings who have been diagnosed with an         inflammatory disease;     -   (ii) suffers from constipation, diarrhea, bloating, urgency,         and/or abdominal pain; and/or     -   (iii) has been administered an antibiotic within the last 1, 2,         or 4 months.

Embodiment 88

The method any one of embodiments 85 to 87, wherein the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

Embodiment 89

The method of any one of embodiments 85 to 88, wherein the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old.

Embodiment 90

The method of any one of embodiments 85 to 88, wherein the subject is between about 2 and about 18 years old, or is at least about 18 years old.

Embodiment 91

The method of any one of embodiments 85 to 90, wherein the subject comprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,         Bifidobacterium spp., and Enterococcus spp. compared to a         healthy or general population;     -   (b) has a reduced proportion of Alternaria alternata,         Aspergillus, flavus, Aspergillus cibarius, and Candida sojae         compared to a healthy or general population;     -   (c) has an increased proportion of Candida albicans and         Debaryomyces spp. compared to a healthy or general population;     -   (d) has a reduced proportion of Bifidobacteria spp.,         Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.         compared to a healthy or general population;     -   (e) has a reduced proportion of Malassezia spp. compared to a         healthy or general population     -   (f) has an increased proportion of Bacterioides spp.,         Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.         compared to a healthy or general population; or     -   (g) has an increased proportion of Enterococcus faecalis,         Enterococcus faecium, or Clostridium difficile compared to a         healthy or general population.

Embodiment 92

The method of any one of embodiments 85 to 91, wherein the biological sample is a bodily fluid.

Embodiment 93

The method of embodiment 92, wherein the bodily fluid is blood, plasma, serum, fecal water, or a brancheoaleolar lavage.

Embodiment 94

The method of embodiment 92 or 93, wherein the bodily fluid is fecal water.

Embodiment 95

The method of any one of embodiments 93 to 94, wherein detecting whether the biological sample is pro-inflammatory comprises contacting an antigen presenting cell with the biological sample.

Embodiment 96

The method of embodiment 95, wherein the antigen presenting cell is a dendritic cell.

Embodiment 97

The method of embodiment 95 or 96, wherein detecting whether the biological sample is pro-inflammatory further comprises contacting a naïve T cell with the antigen presenting cell to produce a contacted T cell.

Embodiment 98

The method of embodiment 97, further comprising detecting a cytokine produced by the contacted T cell and/or the progeny of the contacted T cell.

Embodiment 99

The method of embodiments 97 or 98, wherein biological sample is detected to be pro-inflammatory if

-   -   (i) the proportion of T-helper (TH)-2 cells is increased in the         progeny of the contacted T cell compared to a control;     -   (ii) the proportion of TH-1, TH-17, and/or TH22 cells is         increased in the progeny of the contacted T cell compared to a         control;     -   (iii) the ratio of TH-1 cells to TH-2 cells is decreased in the         progeny of the contacted T cell compared to a control;     -   (iv) the proportion of IL-17 producing CD8+ T cells is increased         in the progeny of the contacted T cell compared to a control;         and/or     -   (v) the amount of IL-4, IL-10, and/or IL-13 produced by the         progeny of the contacted T cell and/or the progeny thereof is         increased compared to a control.

Embodiment 100

The method of embodiment 99, wherein the control is (i) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with sterile culture medium and/or the progeny thereof; (ii) the corresponding proportion, ratio, and/or amount of a corresponding T cell that has been contacted with an antigen presenting cell that has been contacted with a biological sample from a subject who does not have dysbiosis, an inflammatory disease, or a gastrointestinal infection, and/or the progeny thereof; and/or (iii) a reference value corresponding to the proportion, ratio, and/or amount in the general population or a population of subjects who do not have dysbiosis, an inflammatory disease, or a gastrointestinal infection.

Embodiment 101

The method of any one of embodiments 85 to 100, further comprising directing the subject to receive treatment or further testing or monitoring for dysbiosis or an inflammatory disease if the biological sample is detected to be pro-inflammatory.

Embodiment 102

The method of any one of embodiments 85 to 101, further comprising administering the composition of any one of embodiments 52 to 62 to the subject if the biological sample is detected to be pro-inflammatory.

Embodiment 103

The method of any one of embodiments 95 to 102, wherein the subject comprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,         Bifidobacterium spp., and Enterococcus spp. compared to a         healthy or general population;     -   (b) has a reduced proportion of Alternaria alternata,         Aspergillus flavus, Aspergillus cibarius, and Candida sojae         compared to a healthy or general population;     -   (c) has an increased proportion of Candida albicans and         Debaryomyces spp. compared to a healthy or general population;     -   (d) has a reduced proportion of Bifidobacteria spp.,         Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.         compared to a healthy or general population;     -   (e) has a reduced proportion of Malassezia spp. compared to a         healthy or general population     -   (f) has an increased proportion of Bacterioides spp.,         Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.         compared to a healthy or general population; or     -   (g) has an increased proportion of Enterococcus faecalis,         Enterococcus faecium, or Clostridium difficile compared to a         healthy or general population.

Embodiment 104

The method of any one of embodiments 63 to 103, further comprising determining whether the subject has a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,         Bifidobacterium spp., and Enterococcus spp. compared to a         healthy or general population;     -   (b) has a reduced proportion of Alternaria alternata,         Aspergillus flavus, Aspergillus cibarius, and Candida sojae         compared to a healthy or general population;     -   (c) has an increased proportion of Candida albicans and         Debaryomyces spp. compared to a healthy or general population;     -   (d) has a reduced proportion of Bifidobacteria spp.,         Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.         compared to a healthy or general population;     -   (e) has a reduced proportion of Malassezia spp. compared to a         healthy or general population     -   (f) has an increased proportion of Bacterioides spp.,         Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.         compared to a healthy or general population; or     -   (g) has an increased proportion of Enterococcus faecalis,         Enterococcus faecium, or Clostridium difficile compared to a         healthy or general population.

Embodiment 105

A method of treating or preventing dysbiosis or an inflammatory disease in a subject determined to have or be at risk of developing dysbiosis or an inflammatory disease according to the method of any one of embodiments 85 to 103, comprising administering a treatment for dysbiosis or the inflammatory disease to the subject.

Embodiment 106

A method of monitoring the effect of treatment for dysbiosis or an inflammatory disease, the method comprising: (i) obtaining a biological sample from the subject; and (ii) detecting whether the biological sample is pro-inflammatory.

Embodiment 107

A method of determining an inflammatory disease activity in a subject, the method comprising: (i) obtaining a biological sample from the subject; and (ii) detecting whether the biological sample is pro-inflammatory.

Embodiment 108

A method of detecting an anti-inflammatory metabolite in a subject that has or is at risk for developing an inflammatory disease, said method comprising: (i) obtaining a biological sample from said subject; and (ii) determining an expression level of an anti-inflammatory metabolite in said biological sample.

Embodiment 109

A method of determining whether a subject has or is at risk of developing dysbiosis or an inflammatory disease, the method comprising: (i) detecting an expression level of one or more anti-inflammatory metabolites or pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an pro-inflammatory metabolite or a decreased expression level of an anti-inflammatory metabolite relative to the standard control indicates that the subject has or is at risk of developing an inflammatory disease; and (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an inflammatory disease.

Embodiment 110

A method of monitoring the effect of treatment for an inflammatory disease in a subject undergoing inflammatory disease therapy or a patient that has received inflammatory disease therapy comprising: (i) determining a first expression level of an anti-inflammatory or pro-inflammatory metabolite in the subject at a first time point; (ii) determining a second expression level of an anti-inflammatory or pro-inflammatory metabolite in the subject at a second time point; and (iii) comparing the second expression level of an anti-inflammatory or pro-inflammatory metabolite to the first expression level of an anti-inflammatory or pro-inflammatory metabolite, thereby determining the effect of treatment for an inflammatory disease in the subject.

Embodiment 111

A method of determining an inflammatory disease activity in a subject, the method comprising: (i) detecting an expression level of one or more anti-inflammatory or pro-inflammatory metabolites in a subject; (ii) determining whether the expression level is modulated relative to a standard control, thereby determining an inflammatory disease activity in the subject; and (iii) based at least in part on the expression level in step (ii), determining the inflammatory disease activity in the subject.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1. Rationally Designed Microbial Consortium for Gastrointestinal Microbiome Restitution

Without being bound by any scientific theory, Lactobacillus johnsonii shifts the composition of the gut microbiome and increases specific anti-inflammatory fatty acids and carbohydrate metabolites in the gastrointestinal tract. Although some beneficial metabolites are predicted to be microbially produced (e.g., by L. johnsonii and the bacterial species it co-enriches within the gut microbiome), it is also likely that others are host derived in response to an altered gut microbiome. In a study of neonates supplemented daily for the first six months of age with Lactobacillus rhamnosus GG, an altered gut microbiome associated with similar metabolic enrichments persisted for up to 12 months after the cessation of supplementation with Lactobacillus.

Surprisingly, a bacterial population comprising a consortium of bacterial species may be used to prevent or treat chronic inflammatory disease by introducing or restoring the metabolic capacity to regulate inflammatory responses. The consortium of bacterial species (the “consortium”) achieves this by altering microbial colonization patterns in the gastrointestinal tract, and, most importantly, introducing or restoring the capacity to produce a suite of anti-inflammatory metabolites necessary for down-regulation of pro-inflammatory responses. Much of the risk of childhood disease is associated with early life events in microbiological development and this consortium offers the opportunity to treat high-risk neonates and infants.

The consortium may be used as a therapeutic grade formulation or over-the-counter supplement to, e.g., direct appropriate neonatal gut microbiome development and immune maturation. The consortium may also be used as a replacement for fecal transplantation for chronic inflammatory diseases in which member of the consortium are characteristically depleted or as a supplement to direct gut microbiome re-development following perturbation (e.g., peri- or post-antibiotic or anti-microbial administration).

Without being bound by any scientific theory, the species in the exemplary consortium work together with the main anchor probiotic species, L. johnsonii, in a symbiotic manner, with each providing other members of this bacterial guild with nutrients and co-factors for their survival and modulation of host immunity. Intervention using a regimen of microbial consortium (Lactobacillus johnsonii. Akkermansia muciniphila, Faecalibacterium prausnitzii and Myxococcus xanthus), provides improved protection against allergic sensitization due to an effect that is greater than the sum of the effects of the individual consortium members when administered alone. Using a similar mouse model to that previously published (Fujimura et al., (2014). Proc. Natl. Acul. Sci. 111(2) 805-810), an allergic challenge was combined with supplementation of the exemplary microbial consortium. Set forth herein, the host immune response and allergic response was evaluated using histology, qRT-PCR, and flow cytometry.

Cockroach Allergen (CRA) Murine Model.

To investigate the protective effects of supplementation, C57BL/6 mice (7-8 weeks old) were intratracheally sensitized (Day 1-3) and subsequently challenged once a week with cockroach allergen (CRA) for a total of three weeks. The mice were concurrently supplemented with phosphate buffered saline (PBS, negative vehicle control), L. johnsonii (Lj), the microbial consortium lacking L. johnsonii (C-Lj), a complete consortium (C+Lj), or a heat killed complete consortium (C+Lj Heat Killed, control for metabolically inactive consortium). In the first week supplementation was performed daily, followed by supplementation twice a week for the remaining two weeks. All supplementations were performed by oral gavage using bacteria resuspended in 100 μl of PBS. At the conclusion of the study, mice were euthanized, and various tissues (lung, spleen, ileum) were collected for downstream analyses.

Lung Histology.

Lung tissue was collected from each animal and immediately fixed in Carnoy's solution overnight and subsequently dehydrated in 70% ethanol. Three samples from each group were randomly chosen for embedding in paraffin and staining with hematoxylin and eosin (H&E) or Periodic acid-Schiff (PAS). Images for each stained sample were captured using an Aperio Scanscope XT (Leica Biosystems) at 20× magnification. ImageJ was also used to quantify the amount of mucin staining represented in each PAS-stained slide using set threshold parameters in the RGB stack based on the green channel. The percentage of the image that fell within the threshold values was measured and represented the percentage of positive staining within each image analyzed.

qRT-PCR for Gene Expression.

The mRNA from mouse lung was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen). Prior to RNA isolation, lung samples were placed in Lysing Matrix A tubes (MP Bio) with 600 μl of Buffer RLT. Samples were bead-beaten using MPBio FastPrep-24 homogenizer at 5.5 m/s for 30 s. Manufacturer's instructions were followed for the remainder of the RNA isolation procedure. A total of 1.0 μg of RNA per sample was DNase treated and reverse-transcribed using the RT2 First Strand Kit (Qiagen) per the manufacturer's instructions. Quantitative PCR for allergy associated gene expression was performed using the Custom RT Profiler PCR Array (Qiagen) on a QuantStudio 6 Flex System. Reaction conditions were as follows: 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. Gene expression of cytokines was normalized to GAPDH and expressed as fold change compared to gene expression in CRA-challenged PBS-vehicle gavaged mice. Statistical analysis of cytokine expression levels was preformed using Prism 6 software. Gene expression between experimental groups was compared using a Mann-Whitney U test, with p-values≤0.05 considered significant.

CD4⁺ T Cell Isolation and Flow Cytometry Analysis.

Mouse spleens were removed and placed in ice-cold R10 media (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin) (Life Technologies, Carlsbad, Calif.). Tissues were mechanically homogenized using sterile scalpels, followed by collagenase digestion (C6885, Sigma, 1 mg/ml) at 37° C. for 30 minutes in 1:1 R10-PBS solution. The single cell suspensions were obtained by passing digests 10× through a 16-gauge, blunt-end cannula followed by filtrations through a 40 μm filter. Cell suspensions were washed twice with ice cold PBS (2% FCS, 2 mM EDTA) and centrifuged at 1,200 rpm, 4° C., for 10 min to pellet, and resuspended in R10-EDTA media (R10 with 2 mM EDTA) on ice. One million cells were dispensed into each tube for subsequent antibody staining and analysis. Single-cell suspensions of splenocytes from each mouse were aliquoted (1 million cells per well) and subsequently stained with antibodies CD4 (RM4-5, BD Biosciences, Franklin Lakes, N.J.), CD8a(53-6.7, BD), CXCR5(SPRCL5, eBioscience, San Diego, Calif.), PD-1(RMP1-30, BioLegend, San Diego, Calif.), CD25(PC61, BD), and live/dead aqua stain (Life Technologies). Following surface staining, cells were permeabilized using BD Cytofix/Cytoperm and incubated with CD3e(500A2, BD), IFNγ(XMG1.2, BD), IL-4(11B11, BD), IL-17 DEC(eBiol7B7, eBioscience), and FoxP3(FJK-16s, eBioscience) specific antibodies for internal staining. Stained cells were assayed via flow cytometry on a BD LSR II (BD Biosciences).

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism 6 software. Experimental groups were compared by a Kruskal-Wallis test with a Dun's multiple comparison post-test to determine if there were any significant differences between sample groups. In addition, Mann-Whitney tests were used in some cases to directly compare two groups of values. P-values≤0.05 were considered significant.

Results.

Supplementation with the complete consortium (C+Lj) provides the most robust protection against allergic sensitization. Protection is associated with significant decreases in lung mucin secretion (FIGS. 1A-1B), Muc5 gene expression (FIG. 2), and in Th2 cytokine expression (FIGS. 3A-3C). Protection against allergic sensitization by C+Lj is correlated with systemic increases in IL-17 secreting T helper cells (FIG. 4). L. johnsonii is more effective than L. rhamnosus GG and necessary for the attenuation of allergic sensitization associated Muc5ac expression in the lung of CRA challenged C57BL/6 mice (FIG. 5A-5B). The gut microbiota forms a complex functional network that influences both individual microbial members and host immune responses. Rationally designed microbial gastrointestinal consortium provide greater attenuation of allergic airway sensitization than an individual probiotic species.

Example 2. Effects of Consortium Supplementation on a Murine Model of Airway Allergic Sensitization

Without being bound by any scientific theory, the therapeutic consortium (TC) represents a seed microbial guild that aids in the development of a healthy human gut microbiome. A study in C57BL/6 mice was designed, which have a distinct gut microbiome from BALB/c animals and are not protected against allergic airway sensitization following supplementation with L. johnsonii alone, to determine the effects of TC supplementation on allergic airway sensitization.

To investigate the protective effects of TC supplementation C57BL/6 mice were intratracheally sensitized (days 0-2) and subsequently challenged with cockroach allergen (CRA) on days 14 and 20 over the course of a three week period (FIG. 10). The mice were supplemented with either phosphate buffered saline (PBS, negative vehicle control) or the TC on days 0-5, 8, 12, 16, and 19 via oral gavage (FIG. 10). Table 2 and FIG. 17 show treatment groups utilized in this study.

Applicants examined the microbial community composition in the feces of animals in different treatment groups using 16S rRNA sequencing. The community present in that of the TC-supplemented animals was significantly compositionally distinct from that of the control groups (FIG. 11A). Importantly, the TC-supplemented group was enriched for species with the potential for immunomodulatory activity. For example, Bifidobacterium and specific Clostridia species belonging to Clade IV and XIV have been shown to induce T-regulatory cells. In addition, Lachnospira species have been identified as protective against allergic sensitization disease development. Expansion of Bacteroides was characteristic of allergic sensitization in control animals. In conclusion, oral supplementation of mice with the TC promotes increased relative abundance of genera associated with induction of immune tolerance (e.g., Bifidobacteria, Clostridia, Lachnospira and Ruminococcus; FIGS. 11A and 11B).

Oral supplementation with the TC promoted metabolic reprogramming in both the gut lumen and periphery (FIGS. 12A-12B and FIGS. 18A-18C). Increased levels of itaconate, which is associated with a repair macrophage effector phenotype, were also identified in TC supplemented animals.

TC supplemented mice demonstrated significantly reduced allergic inflammation in response to CRA challenge compared with CRA challenged animals treated with PBS (FIGS. 13A-13B; FIGS. 14A-14B; FIGS. 15A-15C). Thus, TC supplementation significantly reduced allergic inflammation in a murine model of airway allergic sensitization.

Oral supplementation of mice with the TC resulted in a repair macrophage effector phenotype (FIGS. 16A-16F). Therefore, TC supplementation is capable of initiating a repair macrophage effector phenotype in a murine model of airway allergic sensitization.

Example 3. In Vitro Assay for Assessment of Immune Activation Status Using Human Fecal Water or Microbial Products

One of the shortcomings of human microbiome studies is the lack of parallel objective immune status information. Provided herein are partner assays for human microbiome studies to determine the extent of immune activation associated with a variety of bodily fluids, such as fecal water or broncheoalveolar lavage fluid, or to assess the capacity of microbial species, or combinations of microbial species to induce immune activation or, conversely induce immune tolerance. The assays provided herein may be used as diagnostics for chronic inflammatory diseases, as well as for screening for bioactive microbial products that induce immune phenotypes associated with disease (and by extension identify target pathways for therapeutic intervention) or represent novel microbial biotherapeutics. No known assay to date has this capacity.

Fecal samples (250 mg) were added to warm PBS (250 μl, containing 20% FCS) at 1 g/1 ml, (w/v), followed with vigorous vortex for 1 minute. Fecal mixtures were incubated for 10 minutes in 37° C., prior to removal of cellular material by microcentrifugation at 14,000 rpm for 5 minutes. Resulting fecal water was sterilized through a 0.2 μm filter and used in DC co-incubations.

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy adult donors by Ficoll-Hypaque gradient centrifugation. DCs were first enriched from the PBMCs using the EasySep™ Human Pan-DC Pre-Enrichment Kit (STEMCELL Technologies, Vancouver, Canada). Enriched DCs (0.5×10⁶ cells/ml) were co-incubated for 48 hours with fecal water (25 μl) and cultured in 96-well plates, in R10 media (RPMI 1640 with 10% heat-inactivated FCS with 2 mM L-glutamine and 100 U/ml penicillin-streptomycin; Life Technologies, Carlsbad, Calif.) supplemented for the first 24 hours with 10 ng/ml GM-CSF and 20 ng/ml IL-4 for. A combination of DC growth factors (10 ng/ml TNF-α, 10 ng/ml IL-1β, 10 ng/ml IL-6, and 1 μM PGE2) were added to the culture for the subsequent 24 hours of incubation. At the end of 48 hour treatment, DCs were washed (once) in fresh media prior to co-culture with CD4+ lymphocytes.

Autologous CD4+ T lymphocytes were purified from PBMC's by negative selection using a CD4+ T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). These isolated T cells were suspended in the TexMACS Medium (Miltenyi Biotec) prior to being added to fecal water exposed DCs at a ratio of 10:1 in the presence of soluble anti-CD28 and anti-CD49d (1 μg/ml). DC and T-cells were co-cultured for 120 hours and replenished with fresh TexMACS Medium every 48 hours. Cells were stimulated with Phorbol Myristate Acetate-lonomycin (Sigma) and GolgiPlug (BD Biosciences) for the final 16 hours of co-incubation. Cell-free media from these co-cultures were collected and evaluated by ELISA (BioLegend) for human cytokines IL-4, IL-10, and IL-13 concentrations.

Single-cell suspensions were stained using two separate panels (phenotype panel and cyctokine panel) of antibodies including BD Biosciences Abs anti-CD3 (SP34-2), anti-CD4 (SK3), anti-CD25 (M-A251), anti-IFNγ (B27), anti-CD8a (RPA-T8, BioLegend), Miltenyi Biotec Abs anti-IL-10 (JES3-9D7), anti-IL-4 (7A3-3), Affymatrix eBioscience Abs anti-IL-22 (22URTI), anti-IL-17A (64DEC17) and anti-FoxP3 (PCH101). Dead cells were identified using LIVE/DEAD® Aqua Dead Cell Stain (Life Technologies). Cells were permeablized by either Cytofix/Cytoperm™ (BD Bioscience) or Fixation/Permeabilization (Affymatrix eBioscience) to stain for intracellular markers, IFNγ, IL-4, IL17A, IL-22, IL-10, FoxP3. Upon flow analysis, live T cells were gated as CD3+CD4+ cells. Amongst the CD4+ T cell sub-populations, Th1 were IFNγ+, Th2 were IL-4+, Th17 cells were IL-17A+, Th22 were IL17A-negative and IL-22+, and T-regulatory cells were both CD25^(hi) and FoxP3^(hi). Stained cells were assayed via flow cytometer on a BD LSR II (BD Biosciences).

Fecal water from a non-atopic neonate significantly reduces CD4+ IL4 and IL13 expression in vitro. Atopy is associated with early-life gastrointestinal bacterial overgrowth and murine microbial metabolism, thus implicating the neonatal gut microbiome in allergic disease development. Microbiota analysis of 298 early-life stool samples from a birth cohort revealed the existence of three compositionally distinct Neonatal Gut Microbiotypes (NGM1, NGM2 and NGM3). NGM3 neonates exhibited significantly higher relative risk for predominantly multisensitized atopy at age two (p<0.03) compared with NGM1 (RR=2.94; 95% CI 1.42-6.09) or NGM 2 (RR=2.06; 95% CI 1.01-4.19), and were more likely to report doctor-diagnosed asthma (p<0.03). Lower-risk NGMs were significantly enriched for commensal bacteria, fungi and a range of luminal anti-inflammatory lipids and carbohydrates. NGM3 neonates exhibited commensal microbial depletion, fungal expansion and metabolic reprogramming manifest as increased pro-inflammatory lipids and host-derived sterols associated with fungal infection.

Findings from this study of neonatal and infant gut microbiomes indicated that neonatal metabolic reprogramming was associated with risk of atopy development at age two and that neonates that exhibited significant increases in anti-inflammatory carbohydrates and lipids in their luminal contents were at significantly decreased risk for allergic sensitization. Previous murine studies have indicated that microbial-derived short chain fatty acids afford protection against airway allergen challenge. Applicants rationalized that fecal water from low-risk neonates (NGM1) which was enriched for known anti-inflammatory lipids and carbohydrates would exhibit the capacity to reduce allergy-associated cytokine expression. Applicants therefore incubated filter-sterilized fecal water from an NGM1 neonate with peripheral blood mononuclear cell-derived dendritic cells isolated from two distinct healthy adult donors, prior to their co-incubation with autologously purified naïve T-cells followed by ionomycin stimulation. Flow cytometry and ELISA analyses, used to examine T-helper 2 (CD4+, IL4+) cells and cytokine production respectively, indicated that fecal water did not substantially influence the number of Th2 cells (FIG. 6A), but significantly and consistently suppressed pro-inflammatory IL4 and IL13 expression (p<0.01 for both; FIG. 6B and FIG. 6C) across both donors.

In vitro DC-T-cell activity assay permits identification of microbes with pro-inflammatory potential. In our studies of the neonatal gut microbiome and atopy, Candida enrichment and host responses to fungal infection (beta-sitosterol and stigmasterol), were amongst the features that characterized high-risk for atopy NGM3 participants. Murine studies employing antimicrobial ablation of the commensal gastrointestinal microbiome, followed by instillation of Candida albicans spores, have previously demonstrated enhanced allergic sensitization even in the absence of allergen exposure. Without wishing to be bound by any scientific theory, it was rationalized that Candida enrichment in the gut microbiome of NMG3 neonates may promote adaptive T-helper cell subsets associated with atopy. Using Candida-selective Sabouraud media, four distinct species were isolated from NGM3 neonatal stool samples and identified using full length ITS sequencing as C. metapsilosis, C. parapsilosis, C. orthopsilosis, and C. tropicalis. Filter-sterilized cell-free supernatant (CFS) from cultures of these four Candida species was used to stimulate peripheral blood mononuclear cell-derived dendritic cells prior to their co-incubation with naïve T-cells in the presence or absence of cockroach antigenic stimulation. Flow cytometry analysis was used to examine T-helper 2 (CD4+, IL4+) and T-regulatory (CD4+, IL10+) subsets. CFS from each of the gastrointestinal Candida species induced significant increases in Th-2 cell proliferation compared to the control (sterile culture medium) exposure, irrespective of cockroach allergen challenge (FIG. 7A). Significant increases were also observed for other pro-inflammatory cytokine producing T-helper cell subsets (TH1, TH17 and TH22). However T-regulatory subsets did not exhibit a consistent significant increase in numbers, indeed most species did not affect T-reg numbers, with one, C. tropicalis, inducing T-regs and another C. metapsilosis inducing a significant reduction in T-reg cell numbers, only in the presence of cockroach allergen stimulation (FIG. 7B). Hence these in vitro analyses corroborate previous animal studies and indicate that the secreted products of distinct Candida species enriched in the gut microbiome of neonates with significantly higher relative risk for atopy at age two have the capacity to drive Th-2 proliferation and cytokine secretion irrespective of antigen presentation by DCs.

In vitro DC/T-cell fecal water assay can be used to discriminate sub-sets of ulcerative colitis patients that exhibit distinct fecal microbiotypes and differ significantly in disease severity. Statistical analyses has permitted us to identify three sub-groups of UC patients based on microbiota composition (bacterial and fungal profiling), referred to MBT-1 to -3). The clinical relevance of gut microbiota-based stratification of our UC patients was assessed by an inter-microbiotype comparison of disease severity (Simple Clinical Colitis Activity (SCCA) index duration (number of years since UC diagnosis), extracolonic manifestations (arthritis, pyoderma gangrenosum, erythema nodosum, and uveitis) and number of first- and second-degree relatives with IBD. MBT-1 patients exhibited higher median SCCA score compared to MBT-2 and MBT-3 groups (FIG. 8A). These patients also exhibited more extracolonic manifestations, and trended towards longer disease duration and a greater number of first- and second-degree relatives diagnosed with IBD (FIGS. 8B-8D).

In an effort to determine the capacity of healthy and disease-associated microbiota to influence adaptive immune responses, we next developed an in vitro assay involving exposure of dendritic cells (DCs; obtained from healthy human donors) to filter-sterilized fecal water from participants in our study, prior to co-culture of exposed DCs with naïve T-cells obtained from the same donor. Flow cytometry was used assess resulting T-cell populations and phenotypes. Compared to healthy control subjects, UC patients were characterized by significant differences in the ratio of CD4+Th1 and Th2 cells; patients exhibited significantly decreased Th1:Th2 ratio (FIG. 9A). Other CD4+ populations (Th17, Th22, and T-regulatory cell) abundances did not exhibit significant differences in relative numbers, nor did CD8+ cell numbers differentiate based on health status. While differences in the Th1:Th2 ratio existed across healthy subjects and UC patients, we postulated that UC-associated gut microbiotypes associated with significantly different disease severity scores would also exhibit concomitant differences in this ratio. We therefore examined cytokine production patterns based on UC-microbiotype. The MBT-1 group, which exhibited the highest disease severity scores exhibited a significantly reduced Th1:Th2 ratio (FIG. 9B), which was associated with an expansion of CD4+, IL4 expressing T-cells (FIG. 9C). Other CD4+ T-cell populations (Th17, Th22, and Treg) also trended towards expansion in MBT-1 group, compared to the MBT2 and MBT3 groups. Additionally, the MBT-1 group exhibited a significant increase in IL-17 producing CD8+ T-cells compared to either MBT-2 and MBT-3 patient samples (FIG. 9D). These in vitro data are consistent with clinical observations in that compared to healthy subjects, UC patients are significantly skewed towards a Th2-enriched population of CD4+ cells, and that UC-microbiotypes exhibit significant differences in both their degree of Th2 skewing and expansion of IL17 producing cytotoxic CD8+ cells. Hence microbiological stratification allows identification of immunologically distinct UC patient populations.

TABLE 2 Treatment groups utilized in murine model of airway allergic sensitization study. Gavage Cockroach Allergen (CRA) Intervention Group −(PBS) PBS Vehicle No CRA + PBS Vehicle CRA + PBS + Therapeutic Consortium CRA + TC (TC) + L. johnsonii (Lj) CRA + Lj + Consortium without Lj (C) CRA + C + Heat-Killed TC (HKTC) CRA + HKTC

TABLE 3 Supplementation with Therapeutic Consortium results in metabolic reprogramming leading to an increase in specific lipid compounds. Compound Compound Type Subtype Compound Lipids Phospholipids 1-palmitoyl-2gamma-linolenoyl- GPC (16:0/18:3n6) Lipids Phospholipids Oleoylcholine Lipids Phospholipids 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6) Lipids Phospholipids 1-palmitoleoyl-2-linoleoyl-GPC (16:1/18:2) Lipids Phospholipids 1-palmitoyl-2-alpha-linolenoyl-GPC (16:0/18:3n3) Lipids Phospholipids 1,2-dioleoyl-GPE (18:1/18:1) Lipids Phospholipids 1-stearoyl-2-linoleoyl-GPE (18:0/18:2) Lipids Phospholipids 1-palmitoyl-2-arachidonoyl-GPE (16:0/20:4) Lipids Phospholipids 1-stearoyl-2-oleoyl-GPE (18:0/18:1) Lipids Phospholipids 1-stearoyl-2-arachidonoyl-GPE (18:0/20:4) Lipids Phospholipids 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1) Lipids Phospholipids 1-stearoyl-2-linoleoyl-GPC (18:0/18:2) Lipids Phospholipids 1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4) Lipids Phospholipids 1,2-dioleoyl-GPC (18:1/18:1) Lipids Phospholipids 1-stearoyl-2-oleoyl-GPC (18:0/18:1) Lipids Phospholipids 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4) Lipids Phospholipids 1-palmitoyl-2-linoleoyl-GPC (16:0/18:2) Lipids Phospholipids 1-palmitoyl-2-oleoyl-GPC (16:0/18:1) Lipids Phospholipids 1,2-dipalmitoyl-GPC (16:0/16:0) Lipids Plasmalogens 1-(1-enyl-oleoyl)-GPE (P-18:1) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-GPE (P-16:0) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2-linoleoyl- GPC (P-16:0/18:2) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2- arachidonoyl-GPC (P-16:0/20:4) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2-oleoyl-GPC (P-16:0/18:1) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2- arachidonoyl-GPE (P-16:0/20:4) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2-linoleoyl- GPE (P-16:0/18:2) Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2-oleoyl-GPE (P-16:0/18:1)

TABLE 4 Supplementation with Therapeutic Consortium results in metabolic reprogramming leading to a decrease in specific carbohydrate, lipid, energy compounds. Compound Type Compound Subtype Compound Carbohydrates Glycolysis/Pyruvate 1,5-anhydroglucitol Carbohydrates Glycolysis/Pyruvate Glucose Carbohydrates Glycolysis/Pyruvate Pyruvate Carbohydrates Glycolysis/Pyruvate Glycerate Carbohydrates Pentose Ribose Carbohydrates Pentose Ribitol Carbohydrates Pentose Ribonate Carbohydrates Pentose Xylose Carbohydrates Pentose Arabinose Carbohydrates Pentose Arabitol/Xylitol Carbohydrates Fructose/Mannose/Galactose Mannitol/Sorbitol Carbohydrates Fructose/Mannose/Galactose Mannose Carbohydrates Fructose/Mannose/Galactose Galactitol (dulcitol) Lipids Polyunsaturated Fatty Acids Docosapentaenoate (n3 DPA; 22:5n3) Lipids Polyunsaturated Fatty Acids Adrenate (22:4n6) Lipids Polyunsaturated Fatty Acids Docosadienoate (22:2n6) Lipids Polyunsaturated Fatty Acids Dihomo-linoleate (20:2n6) Lipids Acyl-glycerols 1-myristoylglycerol (14:0) Lipids Acyl-glycerols 2-myristoylglycerol (14:0) Lipids Acyl-glycerols 1-pentadecanoylglycerol (15:0) Lipids Acyl-glycerols 1-palmitoylglycerol (16:0) Lipids Acyl-glycerols 2-palmitoylglycerol (16:0) Lipids Acyl-glycerols 1-margaroylglycerol (17:0) Lipids Acyl-glycerols 1-oleoylglycerol (18:1) Lipids Acyl-glycerols 2-oleoylglycerol (18:1) Lipids Acyl-glycerols 1-linoleoylglycerol (18:2) Lipids Acyl-glycerols 2-linoleoylglycerol (18:2) Lipids Acyl-glycerols 1-linolenoylglycerol (18:3) Lipids Acyl-glycerols 1-docosahexaenoylglycerol (22:6) Lipids Acyl-glycerols 2-docosahexaenoylglycerol (22:6) Lipids Acyl-glycerols 1-palmitoleoylglycerol (16:1) Lipids Acyl-glycerols 2-palmitoleoylglycerol (16:1) Lipids Acyl-glycerols 1-eicosapentaenoylglycerol (20:5) Lipids Acyl-glycerols 2-eicosapentaenoylglycerol (20:5) Lipids Branched Fatty Acids 15-methylpalmitate Lipids Branched Fatty Acids 17-methylstearate Lipids Branched Fatty Acids 2-hydroxyglutarate Lipids Branched Fatty Acids 1-dihomo-linoleoylglycerol (20:2) Lipids Long Chain Fatty Acids Pentadecanoate (15:0) Lipids Long Chain Fatty Acids Palmitate (16:0) Lipids Long Chain Fatty Acids Margarate (17:0) Lipids Long Chain Fatty Acids 10-heptadecenoate (17:1n7) Lipids Long Chain Fatty Acids Stearate (18:0) Lipids Long Chain Fatty Acids Nonadecanoate (19:0) Lipids Long Chain Fatty Acids 10-nonadecenoate (19:1n9)* Lipids Long Chain Fatty Acids Arachidate (20:0) Lipids Long Chain Fatty Acids Eicosenoate (20:1) Lipids Long Chain Fatty Acids Erucate (22:1n9) Lipids Long Chain Fatty Acids Oleate/Vaccenate (18:1) Energy TCA Cycle Citrate Energy TCA Cycle Succinate Energy TCA Cycle Mesaconate

Example 4. Neonatal Gut Microbiota Associates with Childhood Multisensitized Atopy and T Cell Differentiation

Gut microbiota bacterial depletions and altered metabolic activity at 3 months are implicated in childhood atopy and asthma¹. We hypothesized that compositionally distinct human neonatal gut microbiota (NGM) exist, and are differentially related to relative risk (RR) of childhood atopy and asthma. Using stool samples (n=298; aged 1-11 months) from a US birth cohort and 16S rRNA sequencing, neonates (median age, 35 d) were divisible into three microbiota composition states (NGM1-3). Each incurred a substantially different RR for multisensitized atopy at age 2 years and doctor-diagnosed asthma at age 4 years. The highest risk group, labeled NGM3, showed lower relative abundance of certain bacteria (for example, Bifidobacterium, Akkermansia and Faecalibacterium), higher relative abundance of particular fungi (Candida and Rhodotorula) and a distinct fecal metabolome enriched for pro-inflammatory metabolites. Ex vivo culture of human adult peripheral T cells with sterile fecal water from NGM3 subjects increased the proportion of CD4⁺ cells producing interleukin (IL)-4 and reduced the relative abundance of CD4⁺ CD25⁺FOXP3⁺ cells. 12,13-DiHOME, enriched in NGM3 versus lower-risk NGM states, recapitulated the effect of NGM3 fecal water on relative CD4CD25 forkhead box P3 (FOXP3)⁺ cell abundance. These findings suggest that neonatal gut microbiome dysbiosis might promote CD4⁺ T cell dysfunction associated with childhood atopy.

Atopy, the propensity to produce IgE antibodies in response to allergens, is one of the most common chronic health issues² and is considered to be a substantial risk factor for childhood asthma development³. Recently, the condition has been linked to bacterial taxonomic depletions in the human gut microbiota at 3 months, but not at 12 months, of age¹. We therefore hypothesized that compositionally and functionally distinct neonatal (˜1 month of age) gut microbiota states exist, and that their associated products idiosyncratically influence CD4⁺ populations in a manner that relates to the RR of atopy and asthma development in childhood. We studied independent fecal samples collected during a study visit that targeted 1 month olds (median age 35 d; range 16-138 d; n=130; ‘neonates’) or 6 month olds (median age 201 d; range 170-322 d; n=168; ‘infants’) from participants in the racially and socioeconomically diverse Wayne County Health, Environment, Allergy and Asthma Longitudinal Study birth cohort⁴. Predominantly multisensitized atopy (PM atopy) at age 2 years was defined using latent-class analysis, an unsupervised statistical algorithm that clusters subjects according to their pattern of serum specific-IgE (sIgE) responses to a panel of ten food and aeroallergens⁵ (FIG. 26).

At the population level (independently of atopy status), bacterial community α-diversity (taxon number and distribution) expanded with increasing age (Pearson's correlation, r=0.47, P<0.001). In parallel, fungal α-diversity contracted (Pearson's correlation, r=−0.23, P=0.0014), and a reciprocal relationship between these microbial kingdoms existed (Shannon's index; Pearson's correlation, r=−0.24, P<0.001; FIG. 19). Both bacterial and fungal 3-diversity (interpersonal taxonomic composition) were related to participant age (PERMANOVA; R²=0.056, P<0.001; and R²=0.034, P<0.001, respectively. Neonatal fecal microbiota were typically dominated by Bifidobacteriaceae, Enterobacteriaceae, Malasseziales (Malassezia) and Saccharomycetales (Saccharomyces). Infant participants exhibited sustained presence, but diminished relative abundance, of Bifidobacteriaceae and Enterobacteriaceae, an expansion of Lachnospiraceae (Blautia and Ruminococcus) and fungal communities characteristically dominated by Saccharomycetales (Saccharomyces and Candida), the dominant fungal order in healthy adults⁶. These findings indicate an interkingdom gut microbial co-evolution along an age-associated developmental gradient over the first year of life.

To address our primary hypothesis, a Dirichlet multinomial mixture (DMM) model was used to group participants on the basis of bacterial-community composition⁷; three distinct NGM states (NGM1, 2 and 3) represented the best model fit (FIG. 23). PERMANOVA confirmed that NGM designation explained a small but nontrivial proportion of bacterial β-diversity (PERMANOVA; R²=0.09, P<0.001), indicating that NGMs [which did not differ in age (Kruskal-Wallis; P=0.256; FIG. 20A)] may represent a gradient of microbiota configurations in early-life. NGMs trended toward having a significant relationship with fungal β-diversity (Bray-Curtis; PERMANOVA, R²=0.037, P=0.068), signifying that each NGM co-associates with a mycobiota that varies primarily in the relative abundance of the dominant fungal taxa present. Infant samples were divisible into two compositionally distinct gut microbiota states, IGM1 (typically Bifidobacteriaceae dominated) and IGM2 (typically Lachnospiraceae dominated (unweighted UniFrac; PERMANOVA, R²=0.032, P=0.001)), which differed in age (Wilcoxon rank-sum, P=0.0257); IGM1 participants were younger. IGM states were not related to fungal community β-diversity (Bray-Curtis; PERMANOVA, R²=0.011, P=0.33), presumably because infant subjects were consistently enriched for Saccharomycetales.

According to the conventional definition of atopy (IgE>0.35 IU/ml), no significant difference in RR between NGM groups was observed (FIG. 21). However, when the asthma-predictive⁵ PM atopy definition was used, NGM3 participants incurred a higher RR of atopy at age 2 years, as compared to either NGM1 (RR=2.94, 95% CI 1.42-6.09 P=0.004; FIG. 21) or NGM2 groups (RR=2.06; 95% CI 1.01-4.19, P=0.048; FIG. 21). Even larger effect sizes for NGM3 were observed for RR of parental-reported, doctor-diagnosed asthma at age 4 years (FIG. 21). NGM-associated RR of PM atopy was supported by the sum of specific IgE responses at age 2 years (FIG. 20B). IGM participants did not exhibit different RRs for PM atopy (RR=1.02; 95% CI 0.59-1.75, P=0.94; FIG. 27) or asthma (RR=0.51; 95% CI 0.22-1.17, P=0.11), possibly due to increased age range and microbial heterogeneity within this group. Using available early-life characteristics, we identified factors including season of birth, age at sample collection and breastfeeding to be substantially distinct across IGM states. Detectable dog allergen (Can f 1) concentrations (P=0.045) in the home during the neonatal study visit (lowest in the NGM3 group) and baby gender (NGM3 was almost entirely male) significantly differed across NGMs (P=0.038). Despite adjustment for these and other early-life factors commonly related to allergic disease, the relationship between NGM and atopy or asthma persisted. Only one other large pediatric gut-microbiota atopy study exists¹, the youngest participants of which were substantially older (˜100 d) than the neonates in our cohort (median age, 35 d). The application of our DMM model parameters to this data set identified two compositionally distinct groups (Bifidobacteria-dominated NGM1 and Lachnospiraceae-dominated IGM2; indicating that examination of neonatal stool samples is necessary to identify distinct pioneer microbiota related to differential RR.

NGM3 participants were characteristically depleted of bacterial taxa, including Bifidobacteria (Bifidobacteriaceae), Lactobacillus (Lactobacillaceae), Faecalibacterium (Clostridiaceae) and Akkermansia (Verrucomicrobiaceae), when compared with the NGM1 group (zero-inflated negative binomial regression (ZINB), Benjamini-Hochberg, q<0.05). These observations were consistent when NGM3 was compared to NGM2 and also with previously described atopy-associated taxonomic depletions¹. Mycologically, NGM3 subjects were consistently depleted of multiple Malassezia taxa (ZINB, Benjamini-Hochberg, q<0.20, FIG. 28 and FIG. 29)-striking, given our population-based observation that this genus is characteristically enriched in the neonatal gut microbiota. Fungal taxonomic enrichments in the NGM3 group were also consistent when compared to either of the lower-risk groups, and included Rhodotorula and Candida (FIG. 28 and FIG. 29). Hence, neonatal interkingdom microbiota dysbiosis is characteristic of PM atopy and asthma development in childhood.

NGM3-associated bacterial taxonomic alterations were predicted⁸ to result in a deficiency in amino acid, lipid and xenobiotic metabolism pathways. Untargeted liquid chromatography mass spectrometry identified fecal metabolites present in a subset (n=28) of the representative subjects from each NGM (those with the highest posterior probability of NGM membership). Substantial correlations existed between the 16S rRNA profile, predicted metagenome and the metabolome of NGMs (Procrustes; FIG. 30), indicating a deterministic relationship between bacterial community composition and the metabolic microenvironment of the neonatal gut. Between-group comparisons identified specific metabolites enriched in each NGM (Welch's t test; P<0.05). As previously reported from analysis of the urine of subjects with atopy¹, NGM3 participants exhibited fecal enrichment of primary and secondary bile metabolites. However, more expansive metabolic dysfunction, involving lipid, amino acid, carbohydrate, peptide, xenobiotic, nucleotide, vitamin and energy metabolism pathways—essentially, the bacterial pathways predicted to be deficient in NGM3—was evident. Although the NGM1 and NGM2 groups exhibited distinct metabolic programs, a common subset of metabolites differentiated them from NGM3. These included anti-inflammatory polyunsaturated fatty acids, docosapentaenoate (n3 DPA; 22 n5) and dihomo-γ-linolenate^(9,10) (DGLA; 20:3n3 or n6), succinate and the breast-milk oligosaccharides, 3-fucosyllactose and lacto-N-fucopentaose II, which are known to influence gut epithelial colonization^(11,12). By contrast, NGM3 participants were consistently enriched for 12,13-DiHOME, stigma- and sitosterols, 8-hydroxyoctanoate, α-CEHC and γ-tocopherol.

Sterile fecal water from NGM3 participants (compared to that from NGM1), decreased the ratio of CD4⁺IFNγ⁺:CD4⁺IL-4⁺ cells (linear mixed-effects model (LME), P=0.095; FIG. 24), increased the proportion of CD4⁺IL-4⁺ cells (LME, P<0.001; FIG. 22A) and the concentration of IL-4 released (LME, P=0.045; FIG. 22B) and reduced the percentage of CD4⁺CD25⁺FOXP3⁺ cells (compared with control; LME, P<0.017; FIG. 22C) ex vivo, indicating that the NGM3 gut microenvironment promotes adaptive immune dysfunction associated with established atopic asthma. Weighted correlation network analysis identified 32 metabolic modules, one of which discriminated the three NGMs (ANOVA; P=0.038; FIG. 22D) and contained 12,13-DiHOME, which was identified both as a hub metabolite (highest module membership (MM) value=0.91; FIG. 22E) and most NGM-discriminatory (highest MM to metabolite significance correlation (r=0.86, P<0.001; FIG. 22E). An observation supported by its relative enrichment in NGM3 subjects compared to NGM1 and NGM2 (P<0.05 for both; FIG. 25). All concentrations of 12,13-DiHOME examined reduced the proportion of CD4⁺CD25⁺FOXP3⁺ cells, compared with vehicle treatment (LME, P=0.04, P<0.001, P=0.001 respectively; FIG. 22F).

These findings indicate that neonatal gut microbiota influences susceptibility to childhood allergic asthma, potentially via alterations in the gut microenvironment that influence CD4⁺ T cell populations and function. This suggests that very early-life interventions to manipulate the composition and function of the gut microbiome might offer a viable strategy for disease prevention.

Methods

Accession codes. All sequence data related to this study are available from the European Nucleotide Archive (ENA) under accession number PRJEB13896. Additional information is available in Fujimura et al. 2016.

Study Population. Pregnant women (n=1,258) between the ages of 21 and 49 were recruited from August 2003-November 2007 as part of the Wayne County Health, Environment, Allergy and Asthma Longitudinal Study (WHEALS). WHEALS is a prospective birth cohort from southeastern Michigan designed to investigate early-life risk factors for allergic diseases, as previously described⁴. Briefly, women were considered eligible if they lived in a predefined cluster of contiguous zip codes in and surrounding Detroit, Mich., had no intention of moving out of the area and provided informed written consent. Five follow-up interviews were conducted at 1, 6, 12, 24 and 48 months after the birth of their child, with the 24-month appointment being at a standardized study clinic so that the child could be evaluated by a board-certified allergist. Stool samples were collected from the child at the 1- and 6-month home visits. All aspects of this research were approved by the Henry Ford Hospital Institutional Review Board.

Sample criteria of WHEALS subjects for stool microbiome analyses. For this study, we selected children who had completed their 24-month clinic visit, which included a blood draw for IgE measurements, and had had dust samples collected from their homes at the same time as their stool-sample collection (in =308). Stool samples from children ranging from age 1-11 months were collected from field staff during home visits and stored at −80° C. Samples were randomized before being shipped to the University of California, San Francisco (UCSF), on dry ice, where they were also stored at −80° C. until processed.

PM-atopy and asthma definition. Blood drawn at the 2-year clinic visit was used to determine participants' levels of total and ten allergen-specific IgEs (sIgE): Alternaria (Alternaria alternata), German cockroach (Blattella germanica Bla g 2), dog (Canis lupus familiaris Can f 1), house dust mites (Dermatophagoides farinae Der f 1), hen's egg (egg), cat (Felis domesticus Fel d 1), cow's milk (milk), peanut (Arachis hypogaea), common ragweed (Ambrosia artemisiifolia) and Timothy grass (Phleum pratense). Specific IgEs were measured using the Pharmacia UniCAP system (ThermoFisher Scientific, Waltham, Mass., USA). Latent class analysis was used to group participants into four discrete atopic classes according to sensitization patterns of the ten allergen sIgEs, as with the entire WHEALS cohort⁵. Our subset was assigned to one of four latent classes: (i) Low or no sensitization (in =226); (ii) highly sensitized (both food and inhalant allergens; n=9); (iii) milk- and egg-dominated (n=50) sensitization or (iv) peanut- and inhalant(s)-dominated (n=13) sensitization. Because of the sample size, latent classes ii-iv were collapsed and considered to be “predominately multisensitized” (PM atopy; n=72); remaining subjects represented the “low or no sensitization” group. The conventional definition of atopy (at least one positive test (sIgE≥0.35 IU ml⁻¹) to any of the ten allergens) was also used for comparative purposes. Children were defined as having asthma according to parental-reported doctor diagnosis of asthma at the 4-year interview.

Bacterial- and fungal-community profiling, PICRUSt and metabolomic analyses. DNA extraction. Stool samples from 308 infants were extracted by using a modified cetyltrimethylammonium bromide (CTAB)-buffer-based protocol¹³. Briefly, 0.5 ml of modified CTAB extraction buffer were added to 25 mg of stool in a 2-ml Lysing Matrix E tube (MP Biomedicals, Santa Ana, Calif.) and then incubated (65° C., 15 min). Samples were bead-beaten (5.5 m s⁻¹, 30 s) in a Fastprep-24 (MP Biomedicals, Santa Ana, Calif.), which was followed by the addition of 0.5 ml of phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation (14,000 rpm, 5 min), the supernatant was added to a heavy phase-lock gel tube (5 Prime, Gaithersburg, Md.), and chloroform (v:v) was added. Samples were centrifuged (14,000 rpm, 5 min), and the resulting supernatants were added to fresh tubes, which was followed by the addition of 1 μl of linear acrylamide before PEG-NaCl (2v:v). Samples were incubated (21° C., 2 h), washed with 70% EtOH and resuspended in 10 mM Tris-Cl, pH 8.5.

Sequencing preparation. The V4 region of the 16S rRNA gene was amplified, as designed by Caporaso et al.¹⁴. PCR reactions were performed in 25-μl reactions using 0.025 U Takara Hot Start ExTaq (Takara Mirus Bio Inc, Madison, Wis.), 1× Takara buffer with MgCl₂, 0.4 pmol/μl of F515 and R806 primers, 0.56 mg/ml of bovine serum albumin (BSA; Roche Applied Science, Indianapolis, Ind.), 200 μM of dNTPs and 10 ng of gDNA. Reactions were performed in triplicate with the following: initial denaturation (98° C., 2 min), 30 cycles of 98° C. (20 s), annealing at 50° C. (30 s), extension at 72° C. (45 s) and final extension at 72° C. (10 min). Amplicons were pooled and verified using a 2% TBE agarose e-gel (Life Technologies, Grand Island, N.Y.), before undergoing purification using AMPure SPRI beads (Beckman Coulter, Brea, Calif.), being quality checked with the Bioanalyzer DNA 1000 Kit (Agilent, Santa Clara, Calif.) and being quantified using the Qubit 2.0 Fluorometer and the dsDNA HS Assay Kit (Life Technologies, Grand Island, N.Y.). Samples were pooled and sequenced on the Illumina MiSeq platform, as previously described¹⁵.

The internal transcribed spacer region 2 (ITS2) of the rRNA gene was amplified using the primer pair flTS7 (5′-GTGARTCATCGAATCTTTG-3′) (SEQ ID NO:7) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (SEQ ID NO:8 Primers were designed for the Illumina MiSeq platform, as described above. PCR reactions were performed in triplicate in a 25-μl reaction with 1× Takara buffer (Takara Mirus Bio), 200 nM of each primer, 200 μM dNTPs, 2.75 mM of MgCl₂, 0.56 mg ml⁻¹ of BSA (Roche Applied Science, Indianapolis, Ind.), 0.025 U Takara Hot Start ExTaq and 50 ng of gDNA. Reactions were conducted under the following conditions: initial denaturation (94° C., 5 min), 30 cycles of 94° C. (30 s), annealing at 54° C. (30 s), extension at 72° C. (30 s) and a final extension at 72° C. (7 min). PCR verification and purification were performed as described above. Samples were quantified using KAPA SYBR (KAPA Biosystems, Wilmington, Mass.) qPCR, following the manufacturer's protocol. Samples were pooled in equal moles (50 ng), and prepped and denatured libraries with PhiX spike-in control, as described above, were loaded onto the Illumina MiSeq cartridge.

Sequencing-data processing and quality control. For bacterial sequences, paired-end sequences were assembled using FLASH¹⁶ v.1.2.7 and de-multiplexed by barcode, and low-quality reads (Q score, <30) were discarded in QIIME¹⁷ 1.8. If three consecutive bases were <Q30, then the read was truncated and the resulting read retained in the data set only if it was at least 75% of the original length. Sequences were checked for chimeras using UCHIME¹⁸ and filtered from the data set before operational taxonomic unit (OTU) picking at 97% sequence identification using UCLUST¹⁹ against the Greengenes database²⁰ version 13_5. In embodiments, closely related microorganisms are grouped together based on sequence similarity thresholds (e.g., 97%). OTUs represent a user defined cut off for 16S rRNA sequence identity e.g. 97% identity; all sequences that share at least 97% sequence identity across the sequenced region of the gene form a single OTU. Sequencing reads that failed to cluster with a reference sequence were clustered de novo. Sequences were aligned using PyNAST²¹, and taxonomy was assigned using the RDP classifier and Greengenes reference database version²⁰ 13_5. To de-noise the OTU table, taxa with fewer than five total sequences across all samples were removed. A bacterial phylogenetic tree was built using FastTree²² 2.1.3.

Fungal sequences were quality trimmed (Q score, <25) and adaptor sequences removed using cutadapt²³, after which paired-end reads were assembled with FLASH¹⁶. Sequences were demultiplexed by barcode and truncated to 150 bp before undergoing clustering using USEARCH vers. 7 pipeline, specifically the UPARSE²⁴ function, and being chimera-checked using UCHIME. Taxonomy was assigned using UNITE²⁵ vers. 6.

To normalize variation in read depth across samples, data were rarefied to the minimum read depth of 202,367 sequences per sample for bacteria (n=298) and 30,590 for fungi (n=188). To ensure that a truly representative community was used for analysis for each sample, sequence subsampling at these defined depths was rarefied 100 times. The representative community composition for each sample was defined as that which exhibited the minimum average Euclidean distance to all other OTU vectors generated from all subsamplings for that particular sample. Investigators at UCSF were blinded to sample identity until microbiota data sets underwent the aforementioned processing and were ready for statistical analyses.

Phylogenetic Reconstruction of Unobserved States (PICRUSt).

PICRUSt⁸ was used to predict the pathways of those taxa significantly enriched in each NGM state, according to zero-inflated negative binomial regression and corrected for multiple testing using the Benjamini-Hochberg false-discovery rate²⁶(q<0.05). These taxa were used to generate a new OTU table normalized in PICRUSt, and discriminatory pathways were illustrated in a heat map constructed in R.

Metabolomic Profiling.

Stool samples (200 mg) from each of the three microbiota states, eight NGM3 subjects, and ten from each of NGM1 and NGM2 groups were provided to Metabolon (Durham, N.C.) for ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) using their standard protocol (on the World Wide Web.metabolon.com/). These samples were chosen because they exhibited the highest posterior probability of belonging to a given NGM group and possessed sufficient sample volume for UPLC-MS/MS analysis. Compounds were compared to Metabolon's in-house library of purified standards, which includes more than 3,300 commercially available compounds.

Ex vivo dendritic cell challenge and T cell co-culture. Fecal samples from five of ten NGM1 and seven of eight NGM3 neonates that had undergone metabolic profiling were used (biological replicates). Excluded samples from these groups had insufficient volume for analyses. Fecal samples were homogenized 1 g ml⁻¹ (w:v) in pre-warmed phosphate-buffered saline (PBS) containing 20% FBS (FBS). Samples were vortexed, incubated (37° C., 10 min) and centrifuged (14,000 rpm, 30 min). Supernatant was filter-sterilized through a 0.2-μm filter before being used in the dendritic cell (DC) T cell assay described below. PBS was used as the negative control. Treatment conditions used for the DiHOME experiment included: 75 μM, 130 μM and 200 μM 12,13 DiHOME (Cayman Chemical, Ann Arbor, Mich.) solubilized in 0.4%, 0.15% and 0.05% DMSO, respectively. DiHOME solutions were added to R10 media (Roswell Park Memorial Institute media 1640 with 10% heat-inactivated FBS (antigen activator) and 2 mM 1-glutamine and 100 U ml⁻¹ penicillin-streptomycin added; Life Technologies) and exposed to DCs within 1 h of preparation. Controls included PBS and DMSO (delivered in R10 media) at corresponding percentages used to dissolve the different concentrations of DiHOME. Treatment group size was determined on the basis of preliminary assays that demonstrated the effect size for the suppression of CD4⁺CD25⁺FOXP3⁺ using 130 μM of 12,13 DiHOME was approximately seven, indicating that at least two samples per group were required to achieve a power of >0.80.

Peripheral blood mononuclear cells (PBMCs) were purified from plasma obtained from healthy, de-identified human donors (Blood Centers of the Pacific, San Francisco, Calif.) through the cell-sourcing program that ensures donor confidentiality. Donors signed an agreement acknowledging that their blood may be used for research. PBMCs were isolated using Ficoll-Hypaque gradient centrifugation, washed twice with R10 media and incubated for 18 h. Dendritic cells (DCs) were isolated from PBMCs using the EasySep Human Pan-DC Pre-Enrichment Kit (STEMCELL Technologies, Vancouver, BC). DCs (0.5×10⁶ cells ml⁻¹) from two donors (biological replication) were treated in triplicate (treatment replicate) with either cell-free fecal water (0.22 μM filtered) or varying concentrations of DiHOME, and cultured in R10 media supplemented with 10 ng ml⁻¹ GM-CSF and 20 ng ml⁻¹ IL-4 at 37° C.²⁷ for 2 d, for the fecal-water assay, or 5 d, for the DiHOME experiment. For the DiHOME experiment, freshly prepared media containing DiHOME or control exposures was replaced every 48 h. For the fecal-water experiment, the assay was repeated twice on one donor (technical replicates) and once on donor B owing to insufficient numbers of cells recovered from the latter donor. Treatment replicates were also considered biological replicates because the human donor cells are not clonal.

Twenty-four hours before co-culture with CD4⁺ T cells, DC maturation was stimulated by using DC growth mediators (10 ng ml⁻¹ tumor nuclear factor-α [(TNF-α), 10 ng ml⁻¹ IL-1b, 10 ng ml⁻¹ IL-6 and 1 mM prostaglandin E2 (PGE2)]. In preparation for co-culture, DCs were washed in fresh R10 media, counted via flow cytometry and plated in TexMACs Medium (Miltenyi Biotec, San Diego, Calif.) at 0.5×10⁶ live CD45⁺ cells per well.

Autologous T lymphocytes were purified from the PBMCs using a naïve CD4⁺ T cell isolation kit (Miltenyi Biotec). After purification, naïve autologous CD4⁺ T cells were suspended in the TexMACS Medium (Miltenyi Biotec) and added to the treated DCs at a ratio of 10:1 in the presence of soluble anti-CD28 and anti-CD49d (1 mg ml⁻¹). T and DC cells were co-cultured for 5 d at 37° C. and replenished with fresh TexMACS media every 48 h. To assess cytokine production, the co-cultures were mixed with Phorbol Myristate Acetate-Ionomycin (SIGSa, St. Louis, Mo.) and GolgiPlug (Gplug; BD Biosciences, San Jose, Calif.) for 16 h before flow cytometry. Cell-free media from the co-cultures was collected at 48 h and 5 d, before PMA-Gplug addition, to assess cytokine secretion. Cytokine secretion was evaluated by cytometric bead array, following the manufacturer's protocol (BD Biosciences).

For flow cytometry, single-cell suspensions were stained using a panel of antibodies, including anti-CD3 (SP34-2, 1:100), anti-CD4 (L200, 1:100), anti-CD25 (M-A251, 1:25), anti-IFN-γ (B27, 1:200; BD Biosciences); anti-CD8a (RPA-T8, 1:100; BioLegend, San Diego, Calif.); anti-IL4 (7A3-3, 1:20; Miltenyi Biotec); anti-IL-17A (64DEC17, 1:20) and anti-FOXP3 (PCH101, 1:20; Affymetrix eBioscience, Santa Clara, Calif.). Validation for each primary antibody is provided on the manufacturers' websites. Dead cells were stained positive with LIVE-DEAD Aqua Dead Cell Stain (Life Technologies). Permeabilization buffer (Affymetrix eBioscience) was used to permeabilize cells before staining for the intracellular markers IFN-γ, IL-4, IL-17A and FoxP3. For flow analysis, live T cells were gated as CD3⁺CD4⁺ cells, wells containing <50% live cells were excluded from analyses. Among CD4⁺ T cell subpopulations, T helper 1 (Th1) were IFN-γ, T helper 2 (Th2) were IL-4⁺; T helper 17 (Th17) were IL-17A⁺, and T regulatory (T reg) cells were both CD25^(hi) and FOXP3^(hi). Stained cells were assayed via a flow cytometer on a BD LSR II (BD Biosciences).

Statistical analysis. Shannon's diversity index was calculated using QIIME. Pearson's correlation was used to test for a relationship between bacterial and fungal Shannon's diversity. Distance matrices (unweighted UniFrac²⁸ and Bray-Curtis) were calculated in QIIME to assess compositional dissimilarity between samples, and visualized using PCoA plots constructed in Emperor²⁹. Permutational multivariate analysis of variance (PERMANOVA) was performed using Adonis in the R environment to determine factors that significantly (P<0.05) explained variation in microbiota β-diversity.

To identify clusters of subjects on the basis of bacterial-taxonomy, DMM models were used, which implement an unsupervised Bayesian approach that is based on a Dirichlet prior⁷. The best-fitting DMM model was determined using the Laplace approximation to the negative-log model evidence, testing up to ten underlying microbiota states. Each sample was assigned to a particular neonatal gut microbiota (NGM) state on the basis of the maximum posterior probability of NGM membership. Kruskal-Wallis was used to test whether age differentiated the microbiota states. Relative risk (RR) ratios and corresponding 95% confidence intervals were calculated using PROC GENMOD in SAS version 9.4 (Cary, N.C.). Unadjusted and adjusted RRs were calculated on the basis of log-binomial regression using maximum likelihood estimation or robust Poisson regression, when prevalence ratios were near one or when the log-binomial model did not converge. Two-tailed Welch's t test was used to test whether slgE concentrations (log-transformed) were significantly different between the three NGM states.

To determine which OTUs differed in relative abundance between NGM groups, the zero-inflated negative binomial regression (pscl package) was used as a primary modeling strategy, appropriate for sequence-count data. In cases in which OTU distributions were not zero-inflated and the model failed to converge, the standard negative binomial was used as a secondary modeling strategy. These were corrected for multiple testing using the minimum positive false-discovery rate (q<0.05 for bacteria; q<0.20 for fungi)²⁶. Results were natural-log transformed for illustration on phylogenetic trees using iTOL³⁰ v.3.0. When examining the association between early-life factors and NGMs, P values were calculated on the basis of covariate distribution by ANOVA (numerical, normally distributed), Kruskal-Wallis (numerical, skewed), chi-square (categorical) or Fisher's exact (sparse categorical). Log-binomial-regression model was used to test for confounding factors when assessing the RR of individuals with different microbiota states developing atopy or asthma (PROC GENMOD in SAS version 9.4). Fisher's exact two-tailed test was conducted to test whether breastfeeding was practiced significantly (P<0.05) more often in any particular NGM.

Metabolites exhibiting significantly (P<0.05) different concentrations (log-transformed) between lower-risk NGM states and NGM3 were identified using two-tailed Welch's t test. Shared and distinct super- and sub-pathway products among NGMs were illustrated using Cytoscape, vers. 3.2.1 (ref. 31). Co-occurrence networks of metabolites were constructed using weighted correlation network analysis (WGCNA) with the R package WGCNA to find modules of highly interconnected, mutually exclusive metabolites. Pearson correlations were used to determine intermetabolite relationships, wherein modules are composed of positively correlated metabolites. To avoid spurious modules, the minimum module size was set to five. Module ‘eigenmetabolites’ (referred to as eigengenes) were defined as the first principal component of a given module and considered as a representative measure of the joint metabolic profile of that module. Each eigenmetabolite was used to test (ANOVA) the association between its respective module and NGM, module membership was used to determine the interconnectedness of each metabolite to its assigned module and to identify ‘hub’ metabolites: this was defined as the correlation between each metabolite and the eigenmetabolite (strong positive values indicate high interconnectedness).

Procrustes was used to test for concurrence between communities described by 16S phylogeny, PICRUSt and metabolomics data sets.

To test for T cell and cytokine differences, a linear mixed-effects model (LME) was used (R package ImerTest) and adjusted for donors. Except where indicated, all analyses were conducted in the R statistical programming language.

AOP: Differences in the composition of the gut microbiota of infants associate with relative risk of atopy in childhood, and metabolites linked with these distinct microbial states alter T cell differentiation in vitro.

Issue: Differences in the composition of the gut microbiota of infants associate with relative risk of atopy in childhood, and metabolites linked with these distinct microbial states alter T cell differentiation in vitro.

Gut microbiota-state validation in an independent cohort. To assess the validity of our DMM modeling, the published 16S rRNA data of Arrieta et al.¹ was used (n=319 independent fecal samples collected at approximately 3-12 months of age in the Canadian Healthy Infant Longitudinal Development (CHILD) Study). The specific age of each participant was unavailable and the youngest participants in this cohort were 3 months of age, substantially older than neonates in the WHEALS cohort. Hence the dataset could not be segregated into samples that were > or <6 months of age, as had been performed for our Wayne County Health, Environment, Allergy and Asthma Longitudinal Study (WHEALS) cohort. This limited our capacity to identify neonatal microbiota states associated with subsequent childhood atopy and asthma outcomes. Nonetheless, we used the cohort to determine whether any of the microbiota states identified in our study were replicated in the CHILD cohort. Because of the age range of the CHILD cohort, we applied both our NGM and IGM model parameters to the entire data set. A better model fit (i.e., smaller laplace approximation to the negative log model evidence) was obtained when the CHILD data was fit to the NGM model compared with the IGM model (model fit: 32,502 versus 174,610, respectively) and a two-group solution represented the best fit for the CHILD data. Group 1 (G1) included 221 (69%) participants and group 2 (G2) 98 (31%). The posterior probabilities were on average higher for G1 compared to G2 (0.98 vs. 0.95, respectively). Consistent with our findings, CHILD participants assigned to G1 were typically defined by high Bifidobacteriaceae relative abundance (average relative abundance (aRA): 75%). G2 participants were characterized by Lachnospiraceae (aRA: 39%), Clostridiaceae (aRA: 290%), and Ruminococcaceae (aRA: 12⁰%), more reflective of the IGM2 cluster identified in our cohort.

Code Availability.

The following script may be used to calculate a representative multiply rarefied OTU table from an unrarefied OTU table, an alterative to singly rarefied tables. This approach stabilizes the effect of random sampling and results in an OTU table that is more representative of community composition. Multiple single-rarefied OTU tables are calculated for each sample, and the distance between the subject-specific rarefied vectors calculated. The rarefied vector that is the minimum average (or median) distance from itself to all other rarefied vectors is considered the most representative for that subject and used to represent community composition for that sample in the resulting multiply-rarified OTU table.

library(vegan) library(GUNifFrac)

##Parameters

# specify the raw OTU count table, with samples=rows, taxa=columns # rawtab=otu_tab_t # specify the depth you would like to rarefy your tables to the default is to just use the minimum sequencing #depth raredepth=min(rowSums(rawtab)) # specify the number of rarefied tables you would like to generate to calculate your representative rarefied #table from ntables=100 # specify the distance measure to use to calculate distance between rarefied data sets, for each subject #can be any of the methods available in the vegdist function of vegan distmethod=“euclidean” # specify the method to summarize across distances if mean distance, then summarymeasure=mean #if median distance, then summarymeasure=median # summarymeasure=mean # specify the seed start for the rarefied tables # for each subsequent table, 1 will be added that the previous seed # for reproducibility, always save your seedstart value (or just use the default for simplicity). # seedstart=500 # specify if you want progress updates to be printed # verbose =TRUE## returns a representative rarefied OTU table of class matrix.##functions reprare<-function(rawtab=otu_tab_t, raredepth=min(rowSums(otu_tab_t)), ntables=100, distmethod=euclidean”, summarymeasure=mean, seedstart=500, verbose=TRUE) { raretabs=list( ) for (z in 1:ntables) { if (verbose==TRUE) { print(paste(“calculating rarefied table number”, z, sep=“ ”)) } set.seed(seedstart+z) raretabs[[z]]=Rarefy(rawtab, depth=raredepth)[[1]] } raretabsa=array(unlist(raretabs), dim=c(nrow(raretabs[[z]]), ncol(rawtab), ntables)) final_tab=c( ) for (y in 1:nrow(raretabs[[z]])) { if (verbose==TRUE) { print(paste(“determining rep rarefied vector for subject number”, y, sep=“ ”)) } distmat=as.matrix(vegdist(t(raretabsa[y,,]), method=distmethod)) # distance across reps for subject y distsummary=apply(distmat, 2, summarymeasure) whichbestrep=which(distsummary==min(distsummary))[1] # the best rep is the one with the minimum average/median distance to all other reps. (in case of ties, just select the first) bestrep=raretabsa[y,,whichbestrep] # select that rep only for subject y final_tab=rbind(final_tab, bestrep) # build that rep for subject y into final table } rownames(final_tab)=rownames(raretabs[[z]]) colnames(final_tab)=colnames(rawtab) return(final_tab) } ###### example runs of the function: ###### ### dummy data set for example ### ntaxa=200 nsubj=50 set.seed(444) dummyOTU<-matrix(sample(0:500, ntaxa*nsubj, prob=c(0.7,0.1,0.1,rep(0.1/498, 498)), replace=TRUE), ncol=ntaxa) colnames(dummyOTU)=paste(“OTU”, 1:ntaxa, sep=“ ”) rownames(dummyOTU)=paste(“subj”, 1:nsubj, sep=“ ”) sort(rowSums(dummyOTU)) # sequencing depth is uneven # specify the minimum depth repraretable=reprare(rawtab=dummyOTU, raredepth=min(rowSums(dummyOTU)), ntables=100, distmethod=“euclidean”, summarymeasure=mean, seedstart=500, verbose=TRUE) dim(repraretable) sort(rowSums(repraretable)) # sequencing depth is now even # specify a depth other than the minimum repraretable=reprare(rawtab=dummyOTU, raredepth=3380, ntables=100, distmethod=“euclidean”, summarymeasure=mean, seedstart=500, verbose=TRUE) dim(repraretable) # subjects with less than the minimum are no longer in the table sort(rowSums(repraretable)) # sequencing depth is now even

Example 5: Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients

Significant gut microbiota heterogeneity exists among ulcerative colitis (UC) patients, though the clinical implications of this variance are unknown. We hypothesized that ethnically distinct UC patients exhibit discrete gut microbiotas with unique metabolic programming that differentially influence immune activity and clinical status. Using parallel 16S rRNA and internal transcribed spacer 2 sequencing of fecal samples (UC, 30; healthy, 13), we corroborated previous observations of UC-associated bacterial diversity depletion and demonstrated significant Saccharomycetales expansion as characteristic of UC gut dysbiosis. Furthermore, we identified four distinct microbial community states (MCSs) within our cohort, confirmed their existence in an independent UC cohort, and demonstrated their coassociation with both patient ethnicity and disease severity. Each MCS was uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of the metabolic products of these pathways. Using a novel ex vivo human dendritic cell and T-cell coculture assay, we showed that exposure to fecal water from UC patients caused significant Th2 skewing in CD4⁺ T-cell populations compared to that of healthy participants. In addition, fecal water from patients in whom their MCS was associated with the highest level of disease severity induced the most dramatic Th2 skewing. In embodiments identification of highly resolved UC subsets based on defined microbial gradients or discrete microbial features are exploited for effective therapies.

Despite years of research, the etiology of UC remains enigmatic. Diagnosis is difficult and the patient population heterogeneous, which represents a significant barrier to the development of more effective, tailored therapy. In this study, we demonstrate the clinical utility of the gut microbiome in stratifying UC patients by identifying the existence of four distinct interkingdom pathogenic microbiotas within the UC patient population that are compositionally and metabolically distinct, co-vary with clinical markers of disease severity, and drive discrete CD4⁺ T-cell expansions ex vivo. These findings offer new insight into the potential value of the gut microbiome as a tool for subdividing UC patients, opening avenues to the development of more personalized treatment plans and targeted therapies.

Though murine and human studies support the involvement of the gut microbiota in the development and pathogenesis of ulcerative colitis (UC; a common form of inflammatory bowel disease [IBD]), a single causative microbial agent has not been identified and depletion of bacterial diversity remains the primary constant feature of UC gut microbiome dysbiosis (1). Increasingly, disease endotypes have been described among patients within clinically defined chronic inflammatory diseases (2), suggesting that, in the context of immune dysfunction, distinct pathogenic processes may converge upon a common clinical disorder. Since UC pathogenesis is related to gut microbiome composition, we rationalized that factors that dictate the composition and function of these communities may lead to the development of distinct gut microbiome states that function as discrete pathogenic units to deterministically influence immune activation status and disease severity.

Host genetics, diet, and environmental exposures, three factors encompassed by ethnicity, influence both the gut microbiome and UC pathology (3). Indeed, healthy subjects in the United States, Venezuela, and Malawi exhibit a significant relationship between ethnicity and both the composition and function of the fecal microbiota, with diet representing strong selective pressure on the gut microbial assemblage (4). Independently, Frank et al. demonstrated that in a U.S. cohort, IBD risk alleles ATG16L1 and NOD2 (associated with autophagy and the host response to microbes, respectively) are significantly associated with gut microbiome β diversity (5). However, a meta-analysis of genome-wide association studies indicated that such UC risk alleles characteristic of Caucasian populations do not confer a heightened risk on ethnically distinct north Indian subjects (6). In embodiments, distinct pathogenic microbiotas exist within UC patients that covary with both patient ethnicity and disease severity. In embodiments, these distinct pathogenic microbiotas exhibit a predictable program of luminal metabolism that induces significantly different degrees of Th2 activation.

Results. Interkingdom gut microbiota perturbations are characteristic of UC patients. Our study population consisted of a cohort of 43 subjects (30 UC patients and 13 healthy subjects) of self-reported European or South Asian (SA) ethnicity. Several studies have examined bacterial community composition in fecal samples from UC patients; however, to date, none have examined the mycobiome of adult UC patients. Using parallel, high-resolution bacterial (16S rRNA) and fungal (internal transcribed spacer 2 [ITS2]) biomarker gene profiles, we confirmed that our ethnically restricted UC population exhibited bacterial microbiota dysbiosis consistent with that previously described (1). Compared to healthy subjects, UC patients had significantly reduced a diversity (P=0.010; FIG. 31A) and were compositionally distinct (permutational multivariate analysis of variance [PERMANOVA]: weighted UniFrac, R²=0.058, P=0.023) (FIG. 31B). Neither fungal α- or β-diversity differed between healthy and UC patients (P=0.523; see FIG. 34A) (PERMANOVA: Bray-Curtis, R²=0.038, P=0.129; see FIG. 34B), indicating that while profound bacterial depletion is characteristic of the UC gut microbiota, more subtle changes in fungal taxonomy characterize these patients.

A total of 165 bacterial taxa were significantly differentially enriched in healthy participants and UC patients. Consistent with previous reports, specific Bacteroides and Prevotella species and a number of unclassified members of the families Lachnospiraceae and Ruminococcaceae were among the bacterial taxa most significantly depleted in UC gut microbiotas (8, 9). UC patients also exhibited enrichment of members of the Streptococcus, Bifidobacterium, and Enterococcus genera, which was validated by independent phylogenetic microarray profiling of these same samples and confirms previous reports (8, 9). Only a small number of fungal taxa (n=13) exhibited differential relative abundance. UC patients were depleted of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae while being significantly enriched in Candida albicans and Debaryomyces species. Collectively, these data indicate that the UC-associated gut microbiota is characterized by an interkingdom dysbiosis, highlighted by significant expansion of putatively pathogenic bacterial and fungal species, in the context of depleted bacterial diversity.

UC fecal microbiotas segregate by ethnicity, dominant microbial features, and disease characteristics. We next addressed our hypothesis that ethnicity is associated with distinct interkingdom fecal microbiota in UC patients. Healthy EU and SA participants exhibited no significant difference in bacterial or fungal a diversity (see FIG. 34C and FIG. 34D). However, SA-UC patients consistently exhibited less bacterial diversity than either healthy ethnically matched controls or EU UC patients (see FIG. 34C). They also were significantly depleted of fungal diversity compared to the EU UC group (see FIG. 34D), indicating more severe interkingdom microbiome depletion in these patients, though no difference in clinical disease severity between EU and SA-UC patients was observed (see FIG. 34E). Ethnicity was also significantly associated with bacterial, but not fungal, β diversity when all of the participants were considered (see FIG. 34F and FIG. 34G). Because health status was significantly associated with gut microbial composition (FIG. 31B), it represented a potential confounding factor. We therefore repeated PERMANOVA with only UC patients and showed that, while fungal community composition does not exhibit a significant relationship with patient ethnicity (PERMANOVA: Bray-Curtis, R²=0.061, P=0.107), bacterial β diversity does (PERMANOVA: weighted UniFrac, R²=0.075, P=0.039; FIG. 31C), an observation validated by PhyloChip data (see FIG. 34H). Thus, these data indicate that, despite chronic colonic inflammatory disease, ethnicity remains associated with compositionally distinct bacterial communities in the UC gut, though it explains only a small proportion (7.5%) of the observed variation in β diversity across these patients.

Recent pediatric Crohn's disease studies have demonstrated that patients cluster into subgroups based on patterns of microbial coassociation (10, 11). We next asked whether such patterns exist in our adult UC cohort and relate to patient ethnicity and/or clinical correlates of disease severity. Using hierarchical cluster analysis and multiscale bootstrap resampling, we identified four subgroups of UC patients based on fecal bacterial community composition and termed these microbial community state 1 (MCS1) to MCS4. These distinct patient subgroups were confirmed by PERMANOVA with both 16S rRNA sequence and PhyloChip data (see FIG. 35A and FIG. 35B). MCS distribution differed significantly across ethnicities, with EU UC populations primarily composed of MCS1 and MCS2 while SA UC patients exhibited a relatively equal distribution of all four MCSs (Fisher exact test, P=0.042).

The clinical relevance of grouping patients on the basis of MCSs was assessed by using an intergroup comparison of clinical disease severity (simple clinical colitis activity [SCCA] index) (12), extracolonic manifestations (arthritis, pyoderma gangreno-sum, erythema nodosum, and uveitis), the number of first- and second-degree relatives diagnosed with IBD, and duration (years since UC diagnosis). MCS1 patients exhibited more severe disease with higher median SCCA scores, a significant increase in the number extracolonic manifestations, a greater number of first- and second-degree relatives diagnosed with IBD, and longer disease duration (FIG. 32). Though the number of patients in our study is small, these data provide the first indication that distinct pathogenic UC gut microbiotas exist and are associated with clinical features of disease severity.

UC MCSs exhibit distinct taxonomic enrichments, metag-enomic capacity, and metabolic productivity. The distribution of microbial taxa across the four UC MCSs was assessed to identify specific bacterial and fungal enrichments characteristic of each. Each MCS typically exhibited a distinct dominant bacterial family (MCS1, Bacteroidaceae; MCS2, Lachnospiraceae, Ruminococcaceae; MCS3, Prevotellaceae; MCS4, Bifidobacteriaceae). These MCS-specific bacterial enrichments extended beyond the dominant family and were further emphasized when the highest disease severity group (MCS1) was compared to each of the other three groups (MCS2, -3, or -4). Specifically, a majority of the bacterial taxa enriched in MCS1 were members of the Bacteroides genus, while the other subgroups were enriched for Blautia, Ruminococcus (MCS2), Prevotella (MCS3), or Bifidobacterium (MCS4, generalized linear models, P<0.05) species. Using the dominant bacterial family as a classifier, we validated the existence of MCS1 and −2 (the two major MCSs in EU UC patients) in two publicly available UC microbiota data sets obtained from patients primarily of European descent (9, 11), indicating that these MCSs are not exclusive to our study but exist in UC patient populations nationwide. Mycologically, C. albicans and Debaryomyces species were most highly enriched in MCS1 patients compared to each of the other three MCSs (generalized linear models, P<0.05), indicating that interkingdom gut microbiome expansion of Bacteroides species, C. albicans, and Debaryomyces species is associated with more severe UC disease.

To identify microbiota-derived pathways and products characteristic of each MCS that may modulate the host immune response and contribute to clinical disease severity, we performed in silico metagenomic predictions in parallel with broad-spectrum gas and liquid chromatography mass spectrometry of fecal samples. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt; picrust.github.io/picrust/) (13) was used to predict bacterial functional capacity. Presently, this algorithm cannot be used to predict fungal community function. Predicted metabolic capacity varied significantly by MCS (PERMANOVA: Bray-Curtis, R²=0.384, P=0.002). A total of 144 bacterial KEGG pathways discriminated MCS1 to −4, including those involved in amino acid and lipid biosynthesis and metabolism (Kruskal-Wallis test, q<0.0006). Specifically, differential enrichment of glycerolipid, fatty acid, inositol, and multiple amino acid metabolism pathways, including phenylalanine, tyrosine, tryptophan, glutamate, and glutamine, differentiated these groups. We also generated functional predictions for MCS1 and -2 stool samples from the studies of Morgan et al. and Gevers et al. (9, 11). A total of 121 KEGG pathways were differentially enriched between MCS1 and MCS2 in our study; of these, 74 (61.2%) also discriminated MCS1 from MCS2 in both the data sets of Gevers et al. and Morgan et al., indicating a high degree of conserved microbial function associated with MCS1 and −2 across multiple independent studies.

We hypothesized that the predicted functional differences across MCSs would be manifested as distinct programs of luminal metabolism, particularly since the majority of the pathways that differentiated these communities were involved in amino acid and lipid metabolism. Indeed, each MCS exhibited significantly distinct metabolic programs (PERMANOVA: Canberra, R²=0.209, p=0.004) that were significantly related to both the fecal microbiota present (Mantel test, r=0.38, P<0.0001) and its predicted metagenome (Mantel test, r=0.21, P<0.008). We were particularly interested in those luminal metabolites that discriminated the more severe MCS1 from each of the remaining MCSs. Of the 805 metabolites detected across all of the samples, 207 exhibited significant inter-MCS differences in relative concentration (Welch's t test, P<0.05). Compared to MCS groups with lower disease severity, MCS1, as our in silico predictions suggested, was significantly enriched for ophthalmate (a biomarker of increased oxidative stress and depleted glutathione) (14), oxidative-stress-inducing putrescine (15), proinflammatory p-cresol sulfate (16), 9-hydroxyoctadecadienoic acid and (9-HODE) and 13-HODE (a proinflammatory, leukocyte-recruiting monohydroxy fatty acid) (17, 18), and 9,10-dihydroxyoctadecanoic acid (9,10-DiHOME; a neutrophil-recruiting, cytotoxic dihydroxy fatty acid) (19), as well as bioactive lysolipids involved in leukocyte activation (FIG. 33) (18, 20). In contrast, lower disease severity MCSs (MCS2, -3, and -4) were enriched for a range of potentially protective dipeptides (including anti-inflammatory alanyl-glutamine) (21, 22), γ-glutamyl dipeptides indicative of improved oxidative stress coping mechanisms (23), and antioxidant immu-nosuppressive myo-inositol (24, 25). These observed differences in gut luminal metabolic programming between MCSs associated with high and low UC severities indicate the existence of putative mechanisms to control inflammation in patients with less severe disease.

T-cell activity in vitro is related to MCS and health status. Recent studies have demonstrated that specific gut microbiome-derived metabolites influence Th2 responses (7) and, independently, that proinflammatory cytokine production by T-helper cell populations, including Th2 cells, is a characteristic of UC (26). We therefore hypothesized that the luminal milieus associated with distinct MCSs differentially influence CD4⁺ T-cell activation in a manner consistent with disease severity. To assess this, we developed an ex vive assay involving coincubation of human dendritic cells (DCs; obtained from healthy donors) with filter-sterilized fecal water prepared from study participants' feces. DCs were then cocultured with autologous CD4⁺ T cells prior to analyses of T-cell phenotypes and cytokine productivity. Compared to healthy participants, UC patients exhibited a significant reduction in the ratio of Th1 to Th2 cells, significantly increased numbers of both Th1 and Th17 cells, and trends toward increases in both T-regulatory and Th2 cell populations (linear mixed effects, P<0.05) (FIG. 33A-33E). CD8 T-cell subsets did not differ significantly between healthy participants and UC patients (data not shown). These findings suggest that luminal microbial products captured in sterile fecal water contribute to UC by inducing a Th2-skewed expansion of CD4⁺ T-cell populations.

Having demonstrated the Th2-skewing effect of UC-associated fecal water, we next asked if this immune response varied on the basis of MCS and associated differences in symptom severity, focusing specifically on Th1 and Th2 populations. With the exception of a minor significant increase in Th1 populations in response to MCS4 fecal water, no significant differences in overall Th1 or Th2 cell populations were observed between MCS groups and controls (FIG. 33F and FIG. 33G). However, when the Th1-to-Th2 ratio was calculated for each group, the MCS1 group exclusively exhibited a significantly lower Th1-to-Th2 ratio compared with healthy controls (FIG. 33H). Of note, no difference in the Th1-to-Th2 ratio was observed when UC patients were compared on the basis of ethnicity (EU UC versus SA UC, see FIG. 36), providing evidence that patient ethnicity alone is not responsible for the altered T-cell activity observed ex vivo. Furthermore, when considering the two MCSs demonstrating the greatest difference in disease severity (MCS1 and MCS2), only MCS1 fecal water significantly increased secretion of Th2-associated cytokines compared with healthy controls (FIG. 33I-33K). These ex vivo data provide evidence that compositionally and metabolically distinct UC microbiotas are capable of differentially influencing CD4⁺ T-cell populations in a manner consistent with UC disease severity.

Discussion Heterogeneity among UC patients is poorly understood and represents a significant barrier to more effective therapy. Colitis development necessitates microbial involvement, and gut micro-biome dysbiosis is characteristic of adult UC patients, but while genetic, therapeutic, and environmental factors are related to UC bacterial β diversity, they explain a small proportion of the observed variation in these microbial communities (5, 9). Microbial species engage in inter- and intraspecies interactions that dictate coassociated microbes and their physiology (27, 28). For example, C. albicans coaggregates with specific bacterial species in the oral microbiota, facilitating more robust, stress-resistant mixed-species biofilms (27). In turn, the products of these coassociated bacteria induce a physiological shift toward unicellular morphology in C. albicans (27). Similarly, because of metabolic cross-feeding, Streptococcus gordonii facilitates coassociation with Fusobacterium nucleatum (28). Hence, we rationalized that, under the proinflammatory conditions of the colitic gut, distinct patterns of pathogen coassociation occur whose composition and function are relatively conserved across patients and related to immune activation and disease severity. Our data support the existence of four distinct UC MCSs that differ significantly in their prevalence along ethnic divides. Internal and external validation confirmed the existence of the predominant microbiota states, indicating that, despite inherent patient variability, treatment regimens, and geography, conserved patterns of pathogenic microbiota coassociation exist across UC populations within the United States. To improve our understanding of the progression and development of these MCSs, it will be important for future studies to investigate UC patient factors, be they temporal, clinical, genetic, or environmental, that directly drive the microbiome toward these differential microbial states.

Of the four MCSs identified in our study, MCS1 represented the most ill patient group, implicating the composition and metabolism of MCS1 in enhanced immune activation and increased disease severity. MCS1 characteristically exhibited expansion of Bacteroides species, which can produce enterotoxin previously associated with UC, stimulate interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) secretion in intestinal epithelial cells, and intensify colitis symptoms in a murine model of UC (29-31). MCS1 patients also exhibited the greatest expansion of C. albicans and Debaryomyces species. Gut microbial expansion of these fungal species has also been described in adult and pediatric Crohn's disease, as well as pediatric IBD (Crohn's disease and UC patients combined) (10, 32, 33). Together with our study, these data indicate that expansion of Saccharomycetales fungi in the context of depleted bacterial diversity is a consistent feature of IBD in pediatric and adult populations. Whether C. albicans directly influences UC pathology in patients in our study is unclear. However, gastrointestinal colonization by C. albicans impairs gastrointestinal healing in both UC patients and a murine model of UC and can induce a Th2 response following gastrointestinal infection of mice with antimicrobial-depleted gut microbiota diversity (34, 35).

The MCS2 subgroup was enriched for both Blautia and Rumi-nococcus species, which together may produce anti-inflammatory short-chain fatty acids (36-38). Prevotella species (enriched in MCS3) are capable of suppressing lymphocyte activity, while Bifidobacterium species (enriched in MCS4) can reduce the production of both IL-8 and TNF-α in intestinal epithelial cells (39, 40). It should be noted that one patient in our study, who demonstrated a dramatic enrichment of Porphyromonadaceae (see FIG. 35A and FIG. 35B), was not classified as having one of the four main MCSs identified here and, though removed from our analysis, may represent an additional, clinically relevant MCS that, given additional patient enrollments, future studies may further characterize and draw conclusions from. Though confirmation that the MCSs identified in our study are also present in independent UC microbiome studies indicates the relative durability of these microbial states, their long-term stability cannot be assessed in cross-sectional studies. It is likely that these MCSs represent discrete points along a nonlinear continuum of pathogenic microbial successional states that relate to disease progression and severity, similar to the microbial gradient identified by Gevers et al. in pediatric Crohn's disease (11). Though these cross-sectional studies are informative, more expansive, longitudinal studies are necessary to determine the natural history of the gut microbiome in UC development and progression.

While interkingdom microbial taxonomic states represent an economical means to stratify patients in large studies, the functional capacity and productivity of these compositionally discrete pathogenic microbiota are paramount to dictating host immune responses and clinical disease severity. Indeed, in our study, programs of metabolic productivity idiosyncratic to the predicted pathways encoded by bacteria present in each MCS were identified. In particular, 9-HODE, 13-HODE, 9,10-DiHOME, and lyso-phosphatidylcholines (significantly enriched in MCS1) can increase leukocyte recruitment and proinflammatory cytokine secretion (17-20). Soluble epoxide hydrolase inhibitors, which prevent 9,10-DiHOME formation, attenuate UC in both chemical and genetic murine models (41), underscoring a potential role for these oxylipins as contributors to more severe disease and that treatments inhibiting their production may be especially efficacious in this specific patient subgroup. In addition to enrichment of leukocyte chemotactic metabolites, MCS1 patients also had high fecal concentrations of p-cresol sulfate, a microbe-derived metabolite (42), and putrescine, both of which can stimulate a leukocyte oxidative burst (15, 16). Consistent with these observations, ophthalmate was also enriched in MCS1 patients, indicative of greater oxidative stress due to low or depleted levels of reactive oxygen species (ROS) quenching glutathione (14). While the metabolome of high disease severity MCS1 indicated conditions of high oxidative stress, that of UC MCSs associated with lower disease severity (MCS2 to -4) exhibited an increased capacity for ROS quenching due to enhanced γ-glutamyltransferase activity indicated by enrichment of γ-glutamyl amino acids (critical for maintaining glutathione levels) and high concentrations of superoxide scavenging myo-inositol (23, 24). Metabolic signatures indicative of immunosuppressive activity, such as enrichment of anti-inflammatory dipeptides (i.e., alanyl-glutamine) and myo-inositol (both of which decrease the expression of proinflammatory cytokines and reduce leukocyte recruitment in animal models of colitis) (21, 22, 25), were also observed in MCS2 to −4 with lower disease severity. This suggests that the specific metabolic productivity associated with each MCS may govern host immune activity and resulting differences in UC severity.

MCS-associated luminal products, which include host- and/or microbe-derived immunomodulatory metabolites, provide a multifaceted mechanism by which a pathogenic gut microbiota may influence host physiology and dictate clinical disease severity. Though pathogen-associated molecular patterns (PAMPs) have traditionally been considered paramount to driving host immune responses to microbes, emerging data in the field of immuno-metabolism indicate that microbe-derived metabolites are equally effective in dictating immune cell phenotypes. In addition to the established direct immunomodulatory activity of microbe-derived metabolites such as short-chain fatty acids or p-cresol sulfate (16, 38), recent studies have demonstrated that the gut microbiota-associated metabolites taurine, histamine, and spermine comodulate NLRP6 inflammasome signaling, epithelial IL-18 secretion, and downstream antimicrobial peptide production (43). Indeed, our data suggest that specific programs of microbe-derived metabolism in combination with an array of PAMPs presented by pathogenic bacteria and fungi in the distal gut of UC patients serve as effective drivers of immune dysfunction related to UC disease severity. Support for this concept comes from our demonstration ex vivo that sterile fecal water from the most severely ill MCS1 patients induced the greatest degree of Th2 skewing in T-cell populations and associated cytokine production, a feature not observed among the other subgroups with less severe disease. While this observation does not directly implicate the microbiome as a causative agent of UC, it does provide evidence of the ability of the microbiome to perpetuate the inflammation and symptoms associated with UC in a manner specific to microbiota composition. This finding also indicates that the Th2 skew traditionally considered characteristic of UC patients (26) is not a consistent finding across our cohort and may, in fact, be driven by the most severely ill patients in UC cohorts (i.e., MCS1). Whether or not different inflammatory phenotypes present among UC patients select for phenotype-maintaining microbes or are the result of initial, discrete dysbioses remains to be addressed. Regardless, this raises the possibility that distinct immunological features not examined in this study characterize patients with lower disease activity and distinct gut MCSs. Future larger studies will be important in further characterizing the potential immuno-modulatory contributions of theses MCSs while confirming the observations presented here. Hence, therapies tailored to the specific microbial, metabolic, and immune dysfunctions exhibited by UC patient subgroups may prove a highly efficacious strategy for more effective treatment of this disease.

Materials and Methods. Fecal sample collection and nucleic acid isolation. Stool samples were collected from healthy participants and physician-diagnosed UC patients of either EU or SA ethnicity by using a standardized protocol. Fecal DNA was extracted with a combination of bead beating and the commercially available QIAamp DNA Stool kit (catalog no. 51504; Qiagen, CA).

Bacterial 16S rRNA profiling. Total DNA extracted from fecal samples was used as the template for 16S rRNA gene amplification (in triplicate) with barcoded primers targeting the V4 region as previously described (44). Sequencing libraries were created as previously described (44). Full-length 16S amplicons were also generated and hybridized to the G3 16S rRNA PhyloChip (Affymetrix, CA) as previously described (45).

Fungal ITS2 library preparation. ITS2 sequencing libraries were created with triplicate PCR amplicons per sample.

16S and ITS2 library sequencing Purified sequencing libraries were analyzed with a Bioanalyzer (Agilent), quantified with the Qubit HS ds-DNA Assay kit (Invitrogen), and sequenced with an Illumina MiSeq platform and MiSeq Control Software v2.2.0 according to the manufacturer's instructions (Illumina). FLASH v1.2.7, QIIME 1.8, and usearch software packages were used for sequence read quality filtering, operational taxonomic unit (OTU) picking, and OTU table generation (46-48).

Predicted community metagenome analyses. PICRUSt (picrust.github.io/picrust/) was used to generate in silico bacterial metag-enomes by using 16S rRNA data (13).

Metabolome profiling. To profile fecal metabolites, >200 mg of each frozen stool sample was shipped overnight on dry ice to Metabolon, Inc. (Durham, N.C.), for broad-spectrum gas and liquid chromatography-mass spectrometry.

In vitro DC/T-cell fecal water assay. DCs obtained from anonymous healthy human donors (Blood Centers of the Pacific) were coincubated for 24 h with fecal water prepared from the same fecal samples submitted for metabolite profiling (filter to remove intact cells) prior to stimulation with TNF-α, IL-1f3, IL-6, and prostaglandin E2 and incubated for an additional 24 h to induce maturation. DCs were then harvested, washed, and cocultured with autologous T cells at a ratio of 1/10 for 5 days, with medium replenishment every 2 days. The T-cell phenotype was assessed via flow cytometry, and cytokine secretion was assessed by Cytometric Bead Array analysis (BD Biosciences). The assay was repeated in quadruplicate with distinct donors to ensure that observations were not confounded by the peripheral blood mononuclear cell (PBMC) source.

Statistical analysis. (i) Microbial, metagenomic, and metabolomic analyses. Statistical analyses were performed with QIIME v1.8.0 and the R statistical environment (47, 49). For PhyloChip data, fluorescence intensities were log normalized prior to analysis. (ii) Comparison of clinical measurements of disease severity. Clinical measurements of disease severity were compared between UC MCSs by a Kruskal-Wallis test, followed by a pairwise two-tailed Dunn test. (iii) Analysis of T-cell subsets. A linear mixed-effect model was applied with the lme4 package in R to identify significant differences in the abundance of induced T-cell subpopulations based on sample groups (i.e., UC MCSs) while accounting for potential variation introduced by the PBMC source (i.e., donor) (50).

Microarray and nucleotide sequence data accession numbers. All microarray data have been deposited in the Gene Expression Omnibus database (ncbi.nlm.nih.gov/geo) under accession no. GSE78724. All of the sequence data related to this study are available in the Sequence Read Archive database (ncbi.nlm.nih.gov/sra) under accession no. SRP071201.

Fecal sample collection. Study participants were provided detailed instructions and necessary materials for fecal sample collection. Standardized fecal samples (first stool of the morning) were collect at home by defecating onto a sterile stool collection device (Cat. No. Protocult #120; Ability Building Center, MN ) placed over a toilet seat and using a sterile collection cup with an attached sterile scoop (Cat. No. 80.734.311; Sarstedt, Germany). Following collection, fecal samples were placed in a pre-paid overnight mailer with a frozen ice pack (Cat. No. S-9902; ULINE, CA) and shipped overnight via USPS in accordance with federal regulations. Upon arrival, fecal sample were immediately stored at −80° C. This study was approved by the Committee on Human Research at the University of California, San Francisco (CHR #10-03092). Physician diagnosed Ulcerative Colitis patients (age 18 to 60 years old) were recruited directly from the gastroenterology clinic at UCSF's Mount Zion Campus. A questionnaire was provided to each patient to assess clinical measures of disease severity [Simple Clinical Colitis Activity index (SCCA), extra-colonic manifestations (arthritis, pyoderma gangrenosum, erythema nodosum, and uveitis), number of first- and second-degree relatives diagnosed with IBD, and duration of disease (years since UC diagnosis)]. Healthy volunteers (age 18 to 60 years old) were drawn from patients' families and by word of mouth. All participants were self-reported to be of either European or South Asian ethnicity (FIG. 37). Additionally, all participants resided within a 70-mile radius of San Francisco, Calif. Any participant experiencing pregnancy or breast feeding, severe concomitant disease involving the liver, heart, lungs or kidneys, or antibiotic treatment within the preceding 2 months were excluded from the study.

Fecal DNA isolation. DNA was extracted from individual fecal samples using a combination of bead beating and the commercially available QIAamp® DNA Stool Kit (Cat. No. 51504; QIAGEN, CA). Initially, 1.6 mL of Buffer ASL was added to approximately 100 mg of feces and bead beat for 30 s at 6.0 m/s in a FastPrep-24 instrument (Cat. No. 116004500; MP Biomedicals). Following bead beating, samples were incubated at 95° C. for 5 minutes to improve lysis efficiency of difficult to lyse microbes. The remainder of the DNA isolation was conducted using a QIAcube (Cat. No. 9001292; QIAGEN, CA) according to the QIAamp® DNA Stool Kit Protocol: Isolation of DNA from Stool for Pathogen Detection. Isolated DNA was stored at −80° C. Blank extractions were included as negative controls to monitor for bacterial contamination.

Bacterial 16S rRNA Gene Library Preparation. Bacterial 16S rRNA gene sequencing libraries were created as previously described (52). PCR amplification of the 16S rRNA gene was conducted in triplicate for each sample using barcoded primers targeting the V4 region as previously described (52). Blank extractions were used as template for negative controls to monitor for 16S rRNA contamination. PCR reactions were performed in 25 μl reactions using 0.025 U Takara Hot Start ExTaq (Takara Mirus Bio Inc, Madison, Wis.), 1× Takara buffer with MgCl₂, 0.4 pmol μl⁻¹ of F515 and R806 primers, 0.56 mg ml⁻¹ of bovine serum albumin (BSA; Roche Applied Science, Indianapolis, Ind.), 200 μM of dNTPs, and 10 ng of gDNA. Reactions were performed in triplicate under the following conditions: initial denaturation (98° C. for 2 min) followed by 30 cycles of 98° C. (20 sec), annealing at 50° C. (30 sec), extension at 72° C. (45 sec) and a final extension at 72° C. for 10 min. Following PCR, triplicates were pooled and 16s rRNA amplicon concentrations were determined via gel electrophoresis quantitation. 16S rRNA sequence library was created by pooling all PCR amplicons in equimolar concentrations to a final volume of 75 uL. To remove background, the 16S rRNA sequence library was run on a 2% agarose gel and the 16S amplicon (˜380 bp) was purified using the QIAquick Gel Extraction Kit (Cat. No. 28704; QIAGEN, CA). The 16S rRNA primer sequences are provided in Caporaso J G, Lauber C L, Walters W A, Berg-Lyons D, Huntley J, Fierer N, Owens S M, Betley J, Fraser L, Bauer M, Gormley N, Gilbert J A, Smith G, Knight R. 2012. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621-1624. In embodiments, other primer sequences may be used.

Fungal ITS2 Library Preparation. Fungal internal transcribed spacer 2 (ITS2) sequencing libraries were created using similar methods to those used for the 16S rRNA library. PCR amplification of the ITS2 region was conducted in triplicate for each sample using barcoded primers. PCR reactions were performed in 25 μl reaction with 1× Takara buffer (Takara Mirus Bio), 200 nM of each primer, 200 μM dNTPs, 2.75 mM of MgCl₂, 0.56 mg ml⁻¹ of BSA (Roche Applied Science), 0.025 U Takara Hot Start ExTaq and 50 ng of gDNA. Reactions were conducted under the following conditions: initial denaturation (94° C. for 5 min) followed by 30 cycles of 94° C. (30 sec), annealing at 54° C. (30 sec), extension at 72° C. (30 sec) and a final extension at 72° C. for 7 min. Following PCR, triplicates were pooled and purified using the Agencourt AMPure XP-PCR Purification Kit and associated protocol (Cat. No. A63880, Beckman Coulter). Samples were quantified using the KAPA SYBR FAST qPCR Kit (Cat. No. KK4601, KAPA Biosystems) as recommended by the manufacturers. All purified samples were then pooled in equimolar concentrations based individual sample ITS2 quantification to a final volume of 75 uL.

16S and ITS2 Library Sequencing. Purified sequencing libraries were analyzed using a Bioanalyzer (Aligent), quantified using the Qubit HS dsDNA kit (Invitrogen), and diluted to 2 nM. Diluted sequence libraries were then denatured, diluted to 5.88 pM, and combined with denatured 12.5 pM PhiX spike-in to final concentration of 5 pM. Prepared sequencing libraries were then loaded onto the Illumina MiSeq cartridge (Cat. No. MS-102-3001, Illumina) and sequenced (514 cycles, Read 1: 251 cycles, Index Read: 12 cycles, Read 2: 251 cycles) using a MiSeq platform and MiSeq Control Software v2.2.0 according to the manufacturer's instructions (Illumina). All sequence data related to this study is available in the Sequence Read Archive (SRA) database, ncbi.nlm.nih.gov/sra (accession no. PRJNA313074).

Bacterial 16S rRNA Sequence Processing.

Following paired-end sequencing, paired sequences were assembled using FLASH v1.2.7 with a minimum overlap set at 15 bp (53). Assembled reads were de-multiplexed by barcode and filtered for low quality (Q-score<30) using QIIME 1.8 (54). If the Q-score three consecutive bases were <30, the read was truncated before the low-quality bases. The resulting read was retained in the dataset if it was at least 75% of the original length. Operational taxonomic units (OTUs) were picked at 97% sequence identity using uclust against the GreenGenes 13_8 database (55) (56), retaining OTUs containing >1 sequence read. Reads that failed to hit the reference sequence collection were retained and clustered de novo. Sequences were aligned using PyNAST and taxonomy was assigned using uclust and the GreenGenes 13_8 database (57) (55) (56). PyNAST-aligned sequences were chimera checked using ChimeraSlayer (58), removing putative chimeras and representative sequences that failed PyNAST alignment. A phylogenetic tree was built using FastTree (59). To normalize variation in read depth across samples, data were rarefied to the minimum read depth of 49,518 sequences per sample for bacteria. To ensure that a truly representative community of each sample was used for analysis, sequence sub-sampling at the defined depth was bootstrapped 100 times. The representative community composition for each sample was defined as that which exhibited the minimum average Canberra distance to all other OTU vectors generated from all sub-samplings for that particular sample.

Fungal ITS2 Sequence Processing.

Following paired-end sequencing, paired sequences were assembled using FLASH v1.2.7 with a minimum overlap of 25 bp and a maximum overlap of 290 bp (53). Assembled reads were de-multiplexed by barcode using QIIME 1.8 (54). Assembled reads containing >2 expected errors, as determined by usearch (55), were removed. Singleton reads were removed and OTUs of 97% sequence similarity were generated de novo using usearch8.0 (55). The 8_1_2015 UNITE ITS fungal sequence database and usearch8.0 was used to remove potentially chimeric sequences (60) (55). The ITSx software package was then used to extract the predicted ITS2 region from the reference sequence of non-chimeric OTUs, filtering out OTUs predicted to lack a true ITS2 region in the process (61). Taxonomy was then assigned to non-chimeric. ITS2 extracted OTUs using Bayesian classification with a confidence cut-off of 0.8 in QIIME according to the 8_1_2015 UNITE ITS fungal sequence database (54) (60). OTUs responsible for less that 0.001% of the total sequence reads were removed. To normalize variation in read depth across samples, data were rarefied to the minimum read depth of 6,653 sequences per sample for bacteria. To ensure that a truly representative community of each sample was used for analysis, sequence sub-sampling at the defined depth was bootstrapped 100 times. The representative community composition for each sample was defined as that which exhibited the minimum average Canberra distance to all other OTU vectors generated from all sub-samplings for that particular sample.

Bacterial 16S rRNA Gene Profiling Using PhyloChip. Total DNA extracted from fecal samples was used as template for 16S rRNA gene amplification as previously described (62). PCR amplification was verified on a 1% TBE agarose gel then purified using the QIAquick Gel Extraction kit (Cat. No. 28704. QIAGEN, CA). A total of 500 ng of purified PCR product per sample was then fragmented, biotin-labeled, and hybridized to the G3 16S rRNA PhyloChip (Affymetrix, CA) as previously described (63). Washing, staining, and scanning of arrays were conducted according to standard Affymetrix protocol (63). Background subtraction, detection, taxon quantification criteria and array normalization was performed as previously described (63). Stage 1 thresholds were adjusted, based on quantitative standards to the following: rQ1≥0.25, rQ2≥0.50, rQ3≥0.80. All PhyloChip microarray data reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database, ncbi.nlm.nih.gov/geo (accession no. GSE78724).

Predicted community metagenome analyses. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt; picrust.github.io/picrust/), a bioinformatics software package used to predict functional metagenomes from a marker gene survey (such as 16S rRNA gene), was used to generate in silico bacterial metagenomes for data generated in this study (64). First, the biom-formatted bacterial OTU table previously generated from the processed 16S rRNA gene MiSeq data was filtered to contain only closed-reference OTUs [i.e. OTUs present in the GreenGenes 16S rRNA 13_8 database (56)]. The closed-reference OTU table was then used to generate predicted metagenomes according to the PICRUSt metagenome prediction tutorial (picrust.github.io/picrust/tutorials/metagenome_prediction.html-metagenome-prediction-tutorial). Briefly, OTU abundance was first normalized according to known or predicted 16s copy number. Following 16s copy number normalization, this normalized OTU table was then used to predicted KEGG Ortholog (KO) abundances for each sample, which were further collapsed into KEGG Pathways (genome.jp/kegg/pathway.html).

Metabolome Profiling. To profile fecal metabolites, >200 mg of frozen stool from each sample was shipped overnight on dry ice to Metabolon (Metabolon, NC). Also included were several technical replicate samples created from a homogeneous pool containing a small amount of all study sample. Upon receipt, samples were inventoried, and immediately stored at −80° C. At the time of analysis, samples were extracted and prepared for analysis using Metabolon's standard solvent extraction method (metabolon.com/). The extracted samples were split into equal parts for analysis on the GC/MS and Q-Exactive accurate mass LC/MS platforms.

Sample Preparation:

The sample preparation process was carried out using the automated MicroLab STAR® system from Hamilton Company. Recovery standards were added prior to the first step in the extraction process for QC purposes. Sample preparation was conducted using a proprietary series of organic and aqueous extractions to remove the protein fraction while allowing maximum recovery of small molecules. The resulting extract was divided into two fractions; one for analysis by LC/MS and one for analysis by GC/MS. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. Each sample was then frozen and dried under vacuum. Samples were then prepared for the appropriate instrument, either LC/MS or GC/MS.

QA/QC:

For QA/QC purposes, a number of additional samples were included with each day's analysis. Furthermore, a selection of QC compounds was added to every sample, including those under test. These compounds were chosen so as not to interfere with the measurement of the endogenous compounds. FIG. 38 and FIG. 39 describe the QC samples and compounds. These QC samples are primarily used to evaluate the process control for each study as well as aiding in the data curation.

Ultrahigh Performance Liquid Chromatography Mass Spectroscopy (UPLC/MS/MS):

The LC/MS portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in acidic or basic LC-compatible solvents, each of which contained 8 or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion optimized conditions and the other using basic negative ion optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18-2.1×100 mm, 1.7 μm). Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5 mM Ammonium Bicarbonate. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion, and the scan range was from 80-1000 m/z. Raw data files are archived and extracted as described below.

Gas chromatography Mass Spectrometry (GC/MS):

The samples destined for GC/MS analysis were re-dried under vacuum desiccation for a minimum of 24 hours prior to being derivatized under dried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). The GC column was 5% phenyl/95% dimethyl polysiloxane fused silica column and the temperature ramp was from 40° to 300° C. in a 16 minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron impact ionization. The instrument was tuned and calibrated for mass resolution and mass accuracy on a daily basis. The information output from the raw data files was automatically extracted as discussed below.

Data Extraction and Compound Identification:

Raw data was extracted, peak-identified and QC processed using Metabolon's hardware and software. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards containing the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, nominal mass match to the library +/−0.4 amu, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum.

Normalization:

For studies spanning multiple days, a data normalization step was performed to correct variation resulting from instrument inter-day tuning differences. Essentially, each compound was corrected in run-day blocks by registering the medians to equal one (1.00) and normalizing each data point proportionately. For studies that did not require more than one day of analysis, no normalization was necessary.

In vitro DC/T-cell fecal water assay. Fecal Water Preparation. Fecal samples were diluted in sterile 37° C. PBS containing 20% FBS and 2 mM EDTA to a final concentration of 1 g/mL. Diluted fecal samples were then vortex for 1 minutes and incubated at 37° C. for 10 minutes. Following incubation, samples were centrifuged at ˜21,000 g for 10 minutes at room temperature to remove insoluble material. Supernatants were then filtered through a 0.2 μm nylon filter to remove intact cells. Sterile fecal water solutions were stored at −20° C.

Dendritic Cell Fecal Water Challenge and T-Cell Co-Culture.

Peripheral blood samples were obtained from anonymous healthy human donors (Blood Centers of the Pacific, San Francisco, Calif.). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation (Cat. No. Histopaque-10771; Sigma-Aldrich). Dendritic cells (DCs) were purified from isolated PBMCs using the EasySep™ Human Pan-DC Pre-Enrichment Kit (Cat. No. 19251; STEMCELL Technologies, Canada) and cultured in 96-well plates (0.5×10⁶ cells/ml) in fresh R10 media: RPMI 1640 (Cat. No. 11875; Thermo-Fisher Scientific) supplemented with 10% heat-inactivated FCS (Cat. No. 9871-5244; USA Scientific), 100 U/ml penicillin-streptomycin (Cat. No. 10378016; Life Technologies, CA), 10 ng/ml GM-CSF (Cat. No. 15-GM-010; R&D Systems, MN), and 20 ng/ml IL-4 (Cat. No. 204-IL-010; R&D Systems). Prepared sterile fecal water was added to DC culture at a 1/20 dilution. After a 24 hour incubation, cells were stimulated with 10 ng/ml TNF-α (Cat. No. 300-01A; PeproTech, NJ), 10 ng/ml IL-1β (Cat. No. 200-01B; PeproTech), 10 ng/ml IL-6 (Cat. No. AF-200-06; PeproTech), and 1 μM PGE2 (Cat. No. 72194; STEMCELL Technologies) and incubated for an additional 24 hours to induce DC maturation. T-cells were purified from autologous, monocyte-depleted PBMCs by negative selection using the Human T-Cell Enrichment Column (Cat. No. HTCC-2000; R&D Systems) and were subsequently cultured in TexMACS Medium (Cat. Not. 130-097-196; Miltenyi Biotec, Germany). Following DC stimulation, DCs were harvested, washed, and co-cultured with autologous T-cells at a ratio of 1/10 in the presence of 1 ug/ml soluble anti-CD28 (Cat. No. 555725; BD Biosciences, CA) and 1 μg/ml anti-CD49d (Cat. No. 555501; BD Biosciences) for 5 days, replenishing the media every 2 days. This assay was repeated four times using PBMCs obtained from distinct donors to ensure observations were independent of PBMC source.

Flow Cytometry.

To assess cytokine production, the co-cultures were stimulated with Phorbol Myristate Acetate-lonomycin (Cat. No. 356150010; Fisher Scientific) and GolgiPlug (Cat. No. 555029; BD Biosciences) for 16 hours. Cells were harvested and single-cell suspensions were stained in two separate antibody panels to assess phenotype. Panel 1: anti-CD3 (Cat. No. 557917, BD Biosciences), anti-CD4 (Cat. No. 563028; BD Biosciences), anti-CD8a (Cat. No. 563821; BioLegend), anti-CD25 (Cat. No. 557741; BD Biosciences), anti-FoxP3 (Cat. No. 14-4776-80; eBioscience), and anti-IL10 (Cat. No. 130-096-043; Miltenyi Biotec). Panel 2: anti-CD3 (Cat. No. 557917; BD Biosciences), anti-CD4 (Cat. No. 563028; BD Biosciences), anti-CD8a (Cat. No. 563821; BioLegend), anti-CD69 (Cat. No. 560737; BD Biosciences), anti-INFγ (Cat. No. 560371; BD Biosciences), anti-IL4 (Cat. No. 130-091-647; Miltenyi Biotec), anti-IL117A (Cat. No. 17-7179-42; eBioscience), and anti-IL22 (Cat. No. 25-7229-42; eBioscience). Cells were permeabilized by either Cytofix/Cytoperm™ (Cat. No. 554714; BD Bioscience) or Fixation/Permeabilization (Cat. No. 00-5523-00; Affymatrix eBioscience). Upon staining, live T-cells were gated as CD3⁺CD4⁺ or CD3⁺CD8⁺ cells. Activated T-cells were surface stained CD69hi. Among the CD4⁺ T-cell population, subpopulations were defined as Th1: IFNγ⁺, Th2: IL-4⁺, Th17: IL-17A⁺, Th22: IL17A⁻ and IL-22⁺, and Treg: CD25hi and FoxP3hi. CD8⁺ T-cells subpopulations were defined as Tc1: IFNγ⁺, Tc2: IL-4⁺, and Tc17: IL-17A⁺. Stained cells were assayed via flow cytometry on a BD LSR II (BD Biosciences).

Cytometric Bead Array.

Prior to addition of PMA/Gplug, 100 uL of cell-free supernatant was removed from each co-culture and centrifuged for 1 minute at 3000 rpm. Cytokine secretion was measured using a cytometric bead array (BD Biosciences) and the concentration of IL-4, IL-5, IL-13, and were determined according to the manufacturer's guidelines. Data was acquired by flow cytometry on a BD LSR II (BD Biosciences) and data analysis was performed using the proprietary FCAP Array analysis software (BD Biosciences).

Statistical analysis. Microbial, Metagenomic, and Metabolomic Analysis. Analysis was performed using QIIME v1.8.0 and the R statistical environment (54, 65). Shannon's Diversity and Faith's Phylogenetic Diversity were calculated using QIIME v 1.8.0 and two-tailed t-tests were performed to identify significant between group differences (e.g. UC vs. Healthy) (54). Weighted UniFrac, Canberra, and Bray-Curtis distance matrices were generated using QIIME v 1.8.0 and visualized via NMDS in the R statistical environment using the vegan package (66, 67). For PhyloChip data, fluorescent intensities were log-normalized prior to calculating Canberra distances. Permutational multivariate analysis of variance (PERMANOVA) using calculated distance matrices was used to determine relationships between existing metadata (i.e. Health Status or Ethnicity) and bacterial, fungal, metagenome, or metabolome composition using the adonis function found in vegan (67). Hierarchical cluster analysis combined with multi-scale, bootstrap resampling was performed using the pvclust package in R with 1000 bootstrap replications (68). Correlation between distances matrices was calculated using the mantel function found in vegan (67). To identify significantly enriched or depleted bacterial OTUs, fungal OTUs, and KEGG pathways between relevant sample groups (e.g. UC vs. Healthy), the three-model approach described by Romero et al. was applied (69). Briefly, three linear mixed-effect regression models (negative binomial, zero-inflated negative bionomial, and Poisson) were independently fit to each observation (i.e. OTU or KEGG pathway) and the model with lowest Akaike Information Criterion (AIC) was retained. P-values were computed for only the best-fit models (i.e. those that minimized AIC). To account for false discovery, q-values were calculated based on the computed p-values. For PhyloChip data, significantly enriched or depleted OTUs were determined by applying a two-tailed t-test to log-normalized fluorescent intensities. To identify significantly enriched or depleted fecal metabolites, log-normalized relative concentrations were compared using a Welch's t-test.

Comparison of Clinical Measures of Disease Severity.

Clinical measures of disease severity (i.e. SCCA, number of extra-colonic manifestations, number of diagnosed first- and second-degree relatives, and years since diagnosis were compared between UC-MCS by a Kruskal-Wallis Test followed by pairwise tw-tailed Dun's Test.

Analysis of T-Cell Subsets.

Because the T-cell assay described above was repeated four separate times using PBMCs from four different PBMC donors, a linear mixed effects model was applied using the lme4 package in R to identify significant differences in the abundance of induced T-cell subpopulations based on sample group (i.e. UC-MCS) while accounting for potential variation introduced due to PBMC source (i.e. donor) (70). The following linear mixed effects models were applied to identify changes due to health status (Healthy vs. UC) and UC-MCS (Healthy vs. MCS1, MCS2, MCS3, MCS4) respectively:

Y˜β((EXP_GROUP)+μ(DONOR)+μ(SAMPLE)+ε

Y˜β(MCS)+μ(DONOR)+μ(SAMPLE)+ε

Where Y=a measured, dependent variable such as Th1 abundance, EXP_GROUP=health status (Healthy or UC), MCS=microbial community state (Healthy, MCS1, MCS2, MCS3, or MCS4), DONOR=PBMC donor source (Donor #1 to #4), and SAMPLE=fecal sample study participant.

REFERENCES References for Example 4

-   1. Arrieta, M. C. et al. Early infancy microbial and metabolic     alterations affect risk of childhood asthma. Sci. Transl. Med. 7,     307ra152 (2015). -   2. Asher, M. I., Montefort, S., Bjorksten, B., Lai, C. K. &     Strachan, D. P. W. S. Worldwide time trends in the prevalence of     symptoms of asthma, allergic rhinoconjunctivitis, and eczema in     childhood. Lancet 368, 733-743 (2006). -   3. Simpson, A. et al. Beyond atopy: multiple patterns of     sensitization in relation to asthma in a birth cohort study. Am. J.     Respir. Crit. Care Med. 181, 1200-1206 (2010). -   4. Aichbhaumik, N. et al. Prenatal exposure to household pets     influences fetal immunoglobulin E production. Clin. Exp. Allergy 38,     1787-1794 (2008). -   5. Havstad, S. et al. Atopic phenotypes identified with latent class     analyses at age 2 years. J. Allergy Clin. Immunol. 134, 722-727.e2     (2014). -   6. Hoffmann, C. et al. Archaea and fungi of the human gut     microbiome: correlations with diet and bacterial residents. PLoS One     8, e66019 (2013). -   7. Holmes, I., Harris, K. & Quince, C. Dirichlet multinomial     mixtures: generative models for microbial metagenomics. PLoS One 7,     e30126 (2012). -   8. Langille, M. G. I. et al. Predictive functional profiling of     microbial communities using 16S rRNA marker gene sequences. Nat.     Biotechnol. 31, 814-821 (2013). -   9. Morin, C., Blier, P. U. & Fortin, S. Eicosapentaenoic acid and     docosapentaenoic acid monoglycerides are more potent than     docosahexaenoic acid monoglyceride to resolve inflammation in a     rheumatoid arthritis model. Arthritis Res. Ther. 17, 142 (2015). -   10. Amagai, Y. et al. Dihomo-γ-linolenic acid prevents the     development of atopic dermatitis through prostaglandin DI production     in NC/Tnd mice. J. Dermatol. Sci. 79, 30-37 (2015). -   11. Bode, L. Human milk oligosaccharides: every baby needs a sugar     mama. Glycobiology 22, 1147-1162 (2012). -   12. Weichert, S. et al. Bioengineered 2′-fucosyllactose and     3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and     enteric pathogens to human intestinal and respiratory cell lines.     Nutr. Res. 33, 831-838 (2013). -   13. DeAngelis, K. M. et al. Selective progressive response of soil     microbial community to wild oat roots. ISME J. 3, 168-178 (2009). -   14. Caporaso, J. G. et al Global patterns of 16S rRNA diversity at a     depth of millions of sequences per sample. Proc. Natl. Acad. Sci.     U.S.A. 108 Supplem, 4516-4522 (2011). -   15. Caporaso, J. G. et al. Ultra-high-throughput microbial community     analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6,     1621-1624 (2012). -   16. Magoč, T. & Salzberg, S. L. FLASH: fast length adjustment of     short reads to improve genome assemblies. Bioinformatics 27,     2957-2963 (2011). -   17. Caporaso, J. G. et al. QIIME allows analysis of high-throughput     community sequencing data. Nat. Methods 7, 335-336 (2010). -   18. Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. &     Knight, R. UCHIME improves sensitivity and speed of chimera     detection. Bioinformatics 27, 2194-2200 (2011). -   19. Edgar, R. C. Search and clustering orders of magnitude faster     than BLAST. Bioinformatics 26, 2460-2461 (2010). -   20. McDonald, D. et al. An improved Greengenes taxonomy with     explicit ranks for ecological and evolutionary analyses of bacteria     and archaea. ISME J. 6, 610-618 (2012). -   21. Caporaso, J. G. et al. PyNAST: a flexible tool for aligning     sequences to a template alignment. Bioinformatics 26, 266-267     (2010). -   22. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree     2-approximately maximum-likelihood trees for large alignments. PLoS     One 5, e9490 (2010). -   23. Martin, M. Cutadapt removes adapter sequences from     high-throughput sequencing reads. EMBnet.journal 17, 10-12 (2011). -   24. Edgar, R. C. UPARSE: highly accurate OTU sequences from     microbial amplicon reads. Nat. Methods 10, 996-998 (2013). -   25. Abarenkov, K. et al. The UNITE database for molecular     identification of fungi—recent updates and future perspectives. New     Phytol. 186, 281-285 (2010). -   26. Benjamini, Y. & Hochberg, Y. Controlling the false discovery     rate: A practical and powerful approach to multiple testing. J. Roy.     Stat. Soc. B 57, 289-300 (1995). -   27. Obermaier, B. et al. Development of a new protocol for 2-day     generation of mature dendritic cells from human monocytes. Biol.     Proced. Online 5, 197-203 (2003). -   28. Lozupone, C. & Knight, R. UniFrac: a new phylogenetic method for     comparing microbial communities. Appl. Environ. Microbiol. 71,     8228-8235 (2005). -   29. Vázquez-Baeza, Y., Pirrung, M., Gonzalez, A. & Knight, R.     EMPeror: a tool for visualizing high-throughput microbial community     data. Gigascience 2, 16 (2013). -   30. Letunic, I. & Bork, P. Interactive Tree Of Life v2: online     annotation and display of phylogenetic trees made easy. Nucleic     Acids Res. 39, W475-W478 (2011). -   31. Shannon, P. et al. Cytoscape: A software environment for     integrated models of biomolecular interaction networks cytoscape.     Genome Res. 13, 2498-2504 (2003).

References for Example 5

-   1. Nagalingam N A, Lynch S V. 2012. Role of the microbiota in     inflammatory bowel diseases. Inflamm Bowel Dis 18:968-984. -   2. Wenzel S E. 2012. Asthma phenotypes: the evolution from clinical     to molecular approaches. Nat Med 18:716-725. -   3. Neuman M G, Nanau R M. 2012. Inflammatory bowel disease: role of     diet, microbiota, life style. Transl Res 160:29-44. -   4. Yatsunenko T, Rey F E, Manary M J, Trehan I, Dominguez-Bello M G,     Contreras M, Magris M, Hidalgo G, Baldassano R N, Anokhin A P, Heath     A C, Warner B, Reeder J, Kuczynski J, Caporaso J G, Lozupone C A,     Lauber C, Clemente J C, Knights D, Knight R, Gordon J I. 2012. Human     gut microbiome viewed across age and geography. Nature 486: 222-227. -   5. Frank D N, Robertson C E, Hamm C M, Kpadeh Z, Zhang T, Chen H,     Zhu W, Sartor R B, Boedeker E C, Harpaz N, Pace N R, Li E. 2011.     Disease phenotype and genotype are associated with shifts in     intestinal-associated microbiota in inflammatory bowel diseases.     Inflamm Bowel Dis 17:179-184. -   6. Juyal G, Prasad P, Senapati S, Midha V, Sood A, Amre D, Juyal R     C, BKT. 2011. An investigation of genome-wide studies reported     susceptibility loci for ulcerative colitis shows limited replication     in north Indians. PLoS One 6:e16565. -   7. Trompette A, Gollwitzer E S, Yadava K, Sichelstiel A K, Sprenger     N, Ngom-Bru C, Blanchard C, Junt T, Nicod L P, Harris N L, Marsland     B J. 2014. Gut microbiota metabolism of dietary fiber influences     allergic airway disease and hematopoiesis. Nat Med 20:159-166. -   8. Frank D N, St Amand A L, Feldman R A, Boedeker E C, Harpaz N,     Pace N R. 2007. Molecular-phylogenetic characterization of microbial     community imbalances in human inflammatory bowel diseases. Proc Natl     Acad Sci USA 104:13780-13785. -   9. Morgan X C, Tickle T L, Sokol H, Gevers D, Devaney K L, Ward D V,     Reyes J A, Shah S A, Leleiko N, Snapper S B, Bousvaros A, Korzenik     J, Sands B E, Xavier R J, Huttenhower C. 2012. Dysfunction of the     intestinal microbiome in inflammatory bowel disease and treatment.     Genome Biol 13:R79 -   10. Lewis J D, Chen E Z, Baldassano R N, Otley A R, Griffiths A M,     Lee D, Bittinger K, Bailey A, Friedman E S, Hoffmann C, Albenberg L,     Sinha R, Compher C, Gilroy E, Nessel L, Grant A, Chehoud C, Li H, Wu     G D, Bushman F D. 2015. Inflammation, antibiotics, and diet as     environmental stressors of the gut microbiome in pediatric Crohn's     disease. Cell Host Microbe 18:489-500. -   11. Gevers D, Kugathasan S, Denson L A, Vazquez-Baeza Y, Van Treuren     W, Ren B, Schwager E, Knights D, Song S J, Yassour M, Morgan X C,     Kostic A D, Luo C, Gonzalez A, McDonald D. Haberman Y, Walters T,     Baker S, Rosh J, Stephens M, Heyman M, Markowitz J. Baldassano R,     Griffiths A, Sylvester F, Mack D, Kim S, Crandall W, Hyams J,     Hut-tenhower C, Knight R, Xavier R J. 2014. The treatment-naive     micro-biome in new-onset Crohn's disease. Cell Host Microbe     15:382-392. -   12. Walmsley R S, Ayres R C, Pounder R E, Allan R N. 1998. A simple     clinical colitis activity index. Gut 43:29-32. -   13. Langille M G, Zaneveld J, Caporaso J G, McDonald D, Knights D,     Reyes J A, Clemente J C, Burkepile D E, Vega Thurber R L, Knight R,     Beiko R G, Huttenhower C. 2013. Predictive functional profiling of     microbial communities using 16S rRNA marker gene sequences. Nat     Biotechnol 31: 814-821. -   14. Dello S A, Neis E P, de Jong M C, van Eijk H M, Kicken C H, Olde     Damink S W, Dejong C H. 2013. Systematic review of ophthalmate as a     novel bio-marker of hepatic glutathione depletion. Clin Nutr     32:325-330. -   15. Walters J D, Chapman K J. 1995. Polyamines found in gingival     fluid enhance the secretory and oxidative function of human     polymorphonuclear leukocytes in vitro. J Periodontal Res 30:167-171. -   16. Schepers E, Meert N, Glorieux G, Goeman J, Van der Eycken J,     Vanholder R. 2007. P-cresylsulphate, the main in vivo metabolite of     p-cresol, activates leucocyte free radical production. Nephrol Dial     Transplant 22:592-596. -   17. Henricks P A, Engels F, van der Vliet H, Nijkamp F P. 1991. 9-     and 13-hydroxy-linoleic acid possess chemotactic activity for bovine     and human polymorphonuclear leukocytes. Prostaglandins 41:21-27. -   18. Rolin J, Al-Jaderi Z, Maghazachi A A. 2013. Oxidized lipids and     lysophosphatidylcholine induce the chemotaxis and intracellular     calcium influx in natural killer cells. Immunobiology 218:875-883. -   19. Totani Y, Saito Y, Ishizaki T, Sasaki F, Ameshima S,     Miyamori I. 2000. Leukotoxin and its diol induce neutrophil     chemotaxis through signal transduction different from that of fMLP.     Eur Respir J 15:75-79. -   20. Qin X, Qiu C. Zhao L. 2014. Lysophosphatidylcholine perpetuates     macrophage polarization toward classically activated phenotype in     inflammation. Cell Immunol 289:185-190. -   21. Young D, Ibuki M, Nakamori T, Fan M, Mine Y. 2012. Soy-derived     di- and tripeptides alleviate colon and ileum inflammation in pigs     with dex-tran sodium sulfate-induced colitis. J Nutr 142:363-368. -   22. Hou Y C, Chu C C, Ko T L, Yeh C L, Yeh S L. 2013. Effects of     alanyl-glutamine dipeptide on the expression of colon-inflammatory     mediators during the recovery phase of colitis induced by dextran     sulfate sodium. Eur J Nutr 52:1089-1098. -   23. Mistry D, Stockley R A. 2010. Gamma-glutamyl transferase: the     silent partner? COPD 7:285-290. -   24. Nascimento N R, Lessa L M, Kerntopf M R, Sousa C M, Alves R S,     Queiroz M G. Price J. Heimark D B, Larner J, Du X, Brownlee M, Gow     A, Davis C, Fonteles M C. 2006. Inositols prevent and reverse     endothelial dysfunction in diabetic rat and rabbit vasculature     metabolically and by scavenging superoxide. Proc Natl Acad Sci USA     103:218-223. -   25. Liao J. Seril D N, Yang A L, Lu G G, Yang G Y. 2007. Inhibition     of chronic ulcerative colitis associated adenocarcinoma development     in mice by ino-sitol compounds. Carcinogenesis 28:446-454. -   26. Bamias G, Kaltsa G, Ladas S D. 2011. Cytokines in the     pathogenesis of ulcerative colitis. Discov Med 11:459-467. -   27. Hogan D A, Vik A, Kolter R. 2004. A Pseudomonas aeruginosa     quorum-sensing molecule influences Candida albicans morphology. Mol     Micro-biol 54:1212-1223. -   28. Sakanaka A, Kuboniwa M, Takeuchi H, Hashino E, Amano A. 2015.     Arginine-ornithine antiporter ArcD controls arginine metabolism and     interspecies biofilm development of Streptococcus gordonii. J Biol     Chem 290: 21185-21198. -   29. Prindiville T P, Sheikh R A, Cohen S H, Tang Y J, Cantrell M C,     Silva J. Jr. 2000. Bacteroides fragilis enterotoxin gene sequences     in patients with in-flammatory bowel disease. Emerg Infect Dis     6:171-174. -   30. Ohkusa T, Yoshida T, Sato N, Watanabe S, Tajiri H,     Okayasu I. 2009. Commensal bacteria can enter colonic epithelial     cells and induce proin-flammatory cytokine secretion: a possible     pathogenic mechanism of ulcerative colitis. J Med Microbiol     58:535-545. -   31. Rath H C, Wilson K H, Sartor R B. 1999. Differential induction     of colitis and gastritis in HLA-B27 transgenic rats selectively     colonized with Bacte-roides vulgatus or Escherichia coli. Infect     Immun 67:2969-2974. -   32. Li Q, Wang C, Tang C, He Q, Li N, Li J. 2014. Dysbiosis of gut     fungal microbiota is associated with mucosal inflammation in Crohn's     disease. J Clin Gastroenterol 48:513-523. -   33. Chehoud C, Albenberg L G, Judge C, Hoffmann C, Grunberg S,     Bit-tinger K, Baldassano R N, Lewis J D, Bushman F D, Wu G D. 2015.     Fungal signature in the gut microbiota of pediatric patients with     inflammatory bowel disease. Inflamm Bowel Dis 21:1948-1956. -   34. Zwolinska-Wcislo M, Brzozowski T. Budak A, Kwiecien S, Sliwowski     Z, Drozdowicz D, Trojanowska D, Rudnicka-Sosin L, Mach T, Konturek S     J, Pawlik W W. 2009. Effect of Candida colonization on human     ulcerative colitis and the healing of inflammatory changes of the     colon in the experimental model of colitis ulcerosa. J Physiol     Pharmacol 60:107-118. -   35. Noverr M C, Noggle R M, Toews G B, Huffnagle G B. 2004. Role of     antibiotics and fungal microbiotain driving pulmonary allergic     responses. Infect Immun 72:4996-5003. -   36. Park S-K, Kim M-S, Roh S W, Bae J-W. 2012. Blautia stercoris sp.     nov., isolated from human faeces. Int J Syst Evol Microbiol     62:776-779. -   37. Miller T L, Wolin M J. 1995. Bioconversion of cellulose to     acetate with pure cultures of Ruminococcus albus and a     hydrogen-using acetogen. Appl Environ Microbiol 61:3832-3835. -   38. Park J, Kim M, Kang S G, Jannasch A H, Cooper B, Patterson J,     Kim C H. 2015. Short-chain fatty acids induce both effector and     regulatory T cells by suppression of histone deacetylases and     regulation of the mTOR-S6K pathway. Mucosal Immunol 8:80-93. -   39. Shenker B J, Vitale L, Slots J. 1991. Immunosuppressive effects     of Pre-votella intermedia on in vitro human lymphocyte activation.     Infect Im-mun 59:4583-4589. -   40. Riedel C U, Foata F, Philippe D, Adolfsson O, Eikmanns B J,     Blum S. 2006. Anti-inflammatory effects of bifidobacteria by     inhibition of LPS-induced N F-kappaB activation. World J     Gastroenterol 12:3729-3735. -   41. Zhang W, Li H, Dong H, Liao J, Hammock B D, Yang G Y. 2013.     Soluble epoxide hydrolase deficiency inhibits dextran sulfate     sodium-induced colitis and carcinogenesis in mice. Anticancer Res     33:5261-5271. -   42. Patel K P, Luo F J, Plummer N S, Hostetter T H, Meyer T W. 2012.     The production of p-cresol sulfate and indoxyl sulfate in     vegetarians versus omnivores. Clin J Am Soc Nephrol 7:982-988. -   43. Levy M, Thaiss C A, Zeevi D, Dohnalovi L, Zilberman-Schapira G,     Mahdi J A, David E, Savidor A, Korem T, Herzig Y, Pevsner-Fischer M,     Shapiro H, Christ A, Harmelin A, Halpern Z, Latz E, Flavell R A,     Amit I, Segal E, Elinav E. 2015. Microbiota-modulated metabolites     shape the intestinal microenvironment by regulating NLRP6     inflammasome signaling. Cell 163:1428-1443. -   44. Caporaso J G, Lauber C L, Walters W A, Berg-Lyons D, Huntley J,     Fierer N, Owens S M, Betley J, Fraser L, Bauer M, Gormley N, Gilbert     J A, Smith G, Knight R. 2012. Ultra-high-throughput microbial     community analysis on the Illumina HiSeq and MiSeq platforms. ISME J     6:1621-1624 -   45. Hazen T C, Dubinsky E A, DeSantis T Z, Andersen G L, Piceno Y M,     Singh N, Jansson J K, Probst A, Borglin S E, Fortney J L,     Stringfellow W T, Bill M, Conrad M E, Tom L M, Chavarria K L, Alusi     T R, Lamendella R, Joyner D C, Spier C, Baelum J, Auer M, Zemla M L,     Chakraborty R, Sonnenthal E L, D'Haeseleer P, Holman H Y, Osman S,     Lu Z, Van Nostrand J D, Deng Y, Zhou J, Mason O U. 2010. Deep-sea     oil plume enriches indigenous oil-degrading bacteria. Science     330:204-208. -   46. Magoč T, Salzberg S L. 2011. FLASH: fast length adjustment of     short reads to improve genome assemblies. Bioinformatics     27:2957-2963. -   47. Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman F     D, Costello E K, Fierer N, Peña A G, Goodrich J K, Gordon J I,     Huttley G A, Kelley S T, Knights D, Koenig J E, Ley R E, Lozupone C     A, McDonald D, Muegge B D, Pirrung M, Reeder J, Sevinsky J R,     Turnbaugh P J, Walters W A, Widmann J, Yatsunenko T, Zaneveld J,     Knight R. 2010. QIIME allows analysis of high-throughput community     sequencing data. Nat Methods 7:335-336. -   48. Edgar R C. 2010. Search and clustering orders of magnitude     faster than BLAST. Bioinformatics 26:2460-2461. -   49. R Core Team. 2015. R: a language and environment for statistical     computing. The R Core Team, Vienna, Austria. -   50. Bates D, Machler M, Bolker B, Walker S. 2015. Fitting linear     mixed-effects models using lme4. J Stat Softw 67:1-48. -   51. Walmsley R S, Ayres R C, Pounder R E, Allan R N. 1998. A simple     clinical colitis activity index. Gut 43:29-32. -   52. Caporaso J G, Lauber C L, Walters W A, Berg-Lyons D, Huntley J,     Fierer N, Owens S M, Betley J, Fraser L, Bauer M, Gormley N, Gilbert     J A, Smith G, Knight R. 2012. Ultra-high-throughput microbial     community analysis on the Illumina HiSeq and MiSeq platforms. ISME J     6:1621-1624. -   53. Magoc T, Salzberg S L. 2011. FLASH: fast length adjustment of     short reads to improve genome assemblies. Bioinformatics     27:2957-2963. -   54. Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman F     D, Costello E K, Fierer N, Pena A G, Goodrich J K, Gordon J I,     Huttley G A, Kelley S T, Knights D, Koenig J E, Ley R E, Lozupone C     A, McDonald D, Muegge B D, Pirrung M, Reeder J, Sevinsky J R,     Turnbaugh P J, Walters W A, Widmann J, Yatsunenko T, Zaneveld J,     Knight R. 2010. QIIME allows analysis of high-throughput community     sequencing data. Nat Methods 7:335-336. -   55. Edgar R C. 2010. Search and clustering orders of magnitude     faster than BLAST. Bioinformatics 26:2460-2461. -   56. DeSantis T Z, Hugenholtz P, Larsen N, Rojas M, Brodie E L,     Keller K, Huber T, Dalevi D, Hu P, Andersen G L. 2006. Greengenes, a     chimera-checked 16S rRNA gene database and workbench compatible with     ARB. Appl Environ Microbiol 72:5069-5072. -   57. Caporaso J G, Bittinger K, Bushman F D, DeSantis T Z, Andersen G     L, Knight R. 2010. PyNAST: a flexible tool for aligning sequences to     a template alignment. Bioinformatics 26:266-267. -   58. Haas B J, Gevers D, Earl A M, Feldgarden M, Ward D V, Giannoukos     G, Ciulla D, Tabbaa D, Highlander S K, Sodergren E, Methe B,     DeSantis T Z, Human Microbiome C, Petrosino J F, Knight R, Birren     B W. 2011. Chimeric 16S rRNA sequence formation and detection in     Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21:494-504. -   59. Price M N, Dehal P S, Arkin A P. 2009. FastTree: computing large     minimum evolution trees with profiles instead of a distance matrix.     Mol Biol Evol 26:1641-1650. -   60. Koljalg U, Nilsson R H, Abarenkov K, Tedersoo L, Taylor A F,     Bahram M, Bates S T, Bruns T D, Bengtsson-Palme J, Callaghan T M,     Douglas B, Drenkhan T, Eberhardt U, Duenas M, Grebenc T, Griffith G     W, Hartmann M, Kirk P M, Kohout P, Larsson E, Lindahl B D, Lucking     R, Martin M P, Matheny P B, Nguyen N H, Niskanen T, Oja J, Peay K G,     Peintner U, Peterson M, Poldmaa K, Saag L, Saar I, Schussler A,     Scott J A, Senes C, Smith M E, Suija A, Taylor D L, Telleria M T,     Weiss M, Larsson K H. 2013. Towards a unified paradigm for     sequence-based identification of fungi. Mol Ecol 22:5271-5277. -   61. Bengtsson-Palme J, Ryberg M, Hartmann M, Branco S, Wang Z, Godhe     A, De Wit P. Sánchez-Garcia M, Ebersberger I, de Sousa F, Amend A,     Jumpponen A, Unterseher M, Kristiansson E, Abarenkov K, Bertrand Y J     K, Sanli K, Eriksson K M, Vik U, Veldre V, Nilsson R H. 2013.     Improved software detection and extraction of ITS1 and ITS2 from     ribosomal ITS sequences of fungi and other eukaryotes for analysis     of environmental sequencing data. Methods in Ecology and Evolution     4:914-919. -   62. Cox M J, Allgaier M, Taylor B, Baek M S, Huang Y J, Daly R A,     Karaoz U, Andersen G L, Brown R, Fujimura K E, Wu B, Tran D, Koff J,     Kleinhenz M E, Nielson D, Brodie E L, Lynch S V. 2010. Airway     microbiota and pathogen abundance in age-stratified cystic fibrosis     patients. PLoS One 5:e11044. -   63. Hazen T C, Dubinsky E A, DeSantis T Z, Andersen G L, Piceno Y M,     Singh N, Jansson J K, Probst A, Borglin S E, Fortney J L,     Stringfellow W T, Bill M, Conrad M E, Tom L M, Chavarria K L, Alusi     T R, Lamendella R, Joyner D C, Spier C, Baelum J, Auer M, Zemla M L,     Chakraborty R, Sonnenthal E L, D'Haeseleer P, Holman H Y, Osman S,     Lu Z, Van Nostrand J D, Deng Y, Zhou J, Mason O U. 2010. Deep-sea     oil plume enriches indigenous oil-degrading bacteria. Science     330:204-208. -   64. Langille M G, Zaneveld J. Caporaso J G, McDonald D, Knights D.     Reyes J A, Clemente J C, Burkepile D E, Vega Thurber R L, Knight R,     Beiko R G, Huttenhower C. 2013. Predictive functional profiling of     microbial communities using 16S rRNA marker gene sequences. Nat     Biotechnol 31:814-821. -   65. Team R C. 2015. R: A Language and Environment for Statistical     Computing. -   66. Lozupone C, Knight R. 2005. UniFrac: a new phylogenetic method     for comparing microbial communities. Appl Environ Microbiol     71:8228-8235. -   67. Jari Oksanen F G B, Roeland Kindt, Pierre Legendre, Peter R.     Minchin, R. B. O'Hara, Gavin L. Simpson, Peter Solymos, M. Henry H.     Stevens, and Helene Wagner. 2015. vegan: Community Ecology Package. -   68. Suzuki R, Shimodaira H. 2006. Pvclust: an R package for     assessing the uncertainty in hierarchical clustering. Bioinformatics     22:1540-1542. -   69. Romero R, Hassan S S, Gajer P, Tarca A L, Fadrosh D W, Nikita L,     Galuppi M, Lamont R F, Chaemsaithong P, Miranda J, Chaiworapongsa T,     Ravel J. 2014. The composition and stability of the vaginal     microbiota of normal pregnant women is different from that of     non-pregnant women. Microbiome 2:4. -   70. Bates D. M. M M, Bolker B. M., Walker S. C. 2015. Fitting Linear     Mixed-Effects Models Using {me4. Journal of Statistical Software 67. 

What is claimed is:
 1. A method of treating or preventing dysbiosis in a subject in need thereof, the method comprising administering to the subject an effective amount of a bacterial population comprising at least: Lactobacillus sp., Faecalibacterium sp., and Akkermansia sp.
 2. The method of claim 1, wherein the Lactobacillus sp. is Lactobacillus johnsonii.
 3. The method of claim 1, wherein the Faecalibacterium sp. is Faecalibacterium prausnitzii.
 4. The method of claim 1, wherein the Akkermansia sp. is Akkermansia muciniphila.
 5. The method of claim 1, wherein the Lactobacillus sp. is Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillus parakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillus sucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis; the Faecalibacterium sp., is Faecalibacterium prausnitzii; and the Akkermansia sp. is Akkermansia muciniphila.
 6. The method of claim 1, wherein the bacterial population further comprises Bifidobacterium sp.
 7. The method of claim 6, wherein the Bifidobacterium sp. is Bifidobacterium bifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile.
 8. The method of claim 1, wherein the bacterial population comprises less than about 20 species of bacteria.
 9. The method of claim 1, wherein the bacterial population is part of a bacterial composition that is not a fecal transplant.
 10. The method of claim 1, wherein the bacterial population is part of a bacterial composition that comprises a pharmaceutically acceptable excipient.
 11. The method of claim 1, wherein the bacterial population is administered orally or rectally.
 12. The method of claim 1, wherein the subject has or is suspected of having an inflammatory disease.
 13. The method of claim 12, wherein the inflammatory disease is an allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behcet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, or atopic dermatitis.
 14. The method of claim 13, wherein the inflammatory disease is pediatric allergic asthma or inflammatory bowel disease.
 15. The method of claim 1, wherein the subject has at least 1 relative who has been diagnosed with an inflammatory disease.
 16. The method of claim 1, wherein the subject suffers from constipation, diarrhea, bloating, urgency, or abdominal pain.
 17. The method of claim 1, wherein the subject has been administered an antibiotic within the last 1 month.
 18. The method of claim 1, wherein the subject is a neonate.
 19. The method of claim 1, wherein the subject is less than about 24 months old.
 20. The method of claim 1, wherein the subject is between about 2 and about 18 years old.
 21. The method of claim 1, wherein the subject comprises a gastrointestinal microbiome that (a) has an increased proportion of Streptococcus spp., and Enterococcus spp. compared to a healthy or general population; (b) has a reduced proportion of Alternaria alternata, Aspergillus flavus, Aspergillus cibarius, and Candida sojae compared to a healthy or general population; (c) has an increased proportion of Candida albicans and Debaryomyces spp. compared to a healthy or general population; (d) has a reduced proportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp. compared to a healthy or general population; (e) has a reduced proportion of Malassezia spp. compared to a healthy or general population; (f) has an increased proportion of Bacterioides spp. or Prevotella spp. compared to a healthy or general population; or (g) has an increased proportion of Enterococcus fiaecalis, Enterococcus faecium, or Clostridium difficile compared to a healthy or general population.
 22. The method of claim 1, wherein the effective amount is effective to (i) increase the level of a Bifidobacterium sp., Clostridia sp. belonging to Clade IV or XIV, a Lachnospira sp., and/or a Ruminococcus sp. in the subject; (ii) lower the pH in the feces of the subject; (iii) increase the level of lactic acid in the feces of the subject; (iv) increase the level of circulating itaconate in the subject; (v) treat, reduce, or prevent allergic inflammation in a subject; (vi) reduce an adaptive immune response in an airway of the subject; (vii) reduce dendritic cell activation in a gastrointestinal-associated mesenteric lymoph node; (viii) increase the level of repair macrophages in the lungs, blood, serum, or plasma of the subject; (ix) increase the level of an anti-inflammatory compound in the subject; (x) decrease the level of a pro-inflammatory compound in the subject; (xi) decrease the level of eotaxin expression and/or secretion in the subject; and/or (xii) decrease the level of mucin expression and/or secretion in the subject.
 23. The method of claim 22, wherein the effective amount is effective to decrease the level of mucin secretion and/or secretion in the lungs of the subject.
 24. The method of claim 22, wherein the anti-inflammatory compound is IL-17.
 25. The method of claim 22, wherein the pro-inflammatory compound is IL-4, IL-10, IL-8, IL-13, TNF-α, or MUC5B.
 26. The method of claim 1, further comprising administering a Cystobacter sp. or a fungal microorganism to the subject.
 27. The method of claim 1, wherein the bacterial population is part of a bacterial composition that is lyophilized.
 28. The method of claim 1, wherein the bacterial population is part of a bacterial composition that is in liquid form. 